Methods of treating bacterial infections

ABSTRACT

Methods of treating or ameliorating a bacterial infection comprising administering a composition comprising a cyclic boronic acid ester Compound I in combination with meropenem are disclosed herewith. In some embodiments, the bacterial infection is a lower respiratory tract infection.

INCORPORATION BY REFERENCE TO PRIORITY APPLICATION

The present application claims the benefit of priority to U.S.Provisional Application No. 62/152,668, filed Apr. 24, 2015, which ishereby incorporated by reference in its entirety.

BACKGROUND Field

Embodiments of the present application relate to antimicrobialcompounds, compositions, their use and preparation as therapeuticagents.

Antibiotics have been effective tools in the treatment of infectiousdiseases during the last half-century. From the development ofantibiotic therapy to the late 1980s there was almost complete controlover bacterial infections in developed countries. However, in responseto the pressure of antibiotic usage, multiple resistance mechanisms havebecome widespread and are threatening the clinical utility ofanti-bacterial therapy. The increase in antibiotic resistant strains hasbeen particularly common in major hospitals and care centers. Theconsequences of the increase in resistant strains include highermorbidity and mortality, longer patient hospitalization, and an increasein treatment costs.

Various bacteria have evolved β-lactam deactivating enzymes, namely,β-lactamases, that counter the efficacy of the various β-lactams.β-lactamases can be grouped into 4 classes based on their amino acidsequences, namely, Ambler classes A, B, C, and D. Enzymes in classes A,C, and D include active-site serine β-lactamases, and class B enzymes,which are encountered less frequently, are Zn-dependent. These enzymescatalyze the chemical degradation of β-lactam antibiotics, renderingthem inactive. Some β-lactamases can be transferred within and betweenvarious bacterial strains and species. The rapid spread of bacterialresistance and the evolution of multi-resistant strains severely limitsβ-lactam treatment options available.

The increase of class D β-lactamase-expressing bacterium strains such asAcinetobacter baumannii has become an emerging multidrug-resistantthreat. A. baumannii strains express A, C, and D class β-lactamases. Theclass D β-lactamases such as the OXA families are particularly effectiveat destroying carbapenem type β-lactam antibiotics, e.g., imipenem, theactive carbapenems component of Merck's Primaxin® (Montefour, K.; et al.Crit. Care Nurse 2008, 28, 15; Perez, F. et al. Expert Rev. Anti Infect.Ther. 2008, 6, 269; Bou, G.; Martinez-Beltran, J. Antimicrob. AgentsChemother. 2000, 40, 428. 2006, 50, 2280; Bou, G. et al, J. Antimicrob.Agents Chemother. 2000, 44, 1556). This has imposed a pressing threat tothe effective use of drugs in that category to treat and preventbacterial infections. Indeed the number of catalogued serine-basedβ-lactamases has exploded from less than ten in the 1970s to over 300variants. These issues fostered the development of five “generations” ofcephalosporins. When initially released into clinical practice,extended-spectrum cephalosporins resisted hydrolysis by the prevalentclass A β-lactamases, TEM-1 and SHV-1. However, the development ofresistant strains by the evolution of single amino acid substitutions inTEM-1 and SHV-1 resulted in the emergence of the extended-spectrumβ-lactamase (ESBL) phenotype.

New β-lactamases have recently evolved that hydrolyze the carbapenemclass of antimicrobials, including imipenem, biapenem, doripenem,meropenem, and ertapenem, as well as other β-lactam antibiotics. Thesecarbapenemases belong to molecular classes A, B, and D. Class Acarbapenemases of the KPC-type predominantly in Klebsiella pneumoniaebut now also reported in other Enterobacteriaceae, Pseudomonasaeruginosa and Acinetobacter baumannii. The KPC carbapenemase was firstdescribed in 1996 in North Carolina, but since then has disseminatedwidely in the US. It has been particularly problematic in the New YorkCity area, where several reports of spread within major hospitals andpatient morbidity have been reported. These enzymes have also beenrecently reported in France, Greece, Sweden, United Kingdom, and anoutbreak in Germany has recently been reported. Treatment of resistantstrains with carbapenems can be associated with poor outcomes.

Another mechanism of β-lactamase mediated resistance to carbapenemsinvolves combination of permeability or efflux mechanisms combined withhyper production of beta-lactamases. One example is the loss of a porincombined in hyperproduction of ampC beta-lactamase results in resistanceto imipenem in Pseudomonas aeruginosa. Efflux pump over expressioncombined with hyperproduction of the ampC β-lactamase can also result inresistance to a carbapenem such as meropenem.

Because there are three major molecular classes of serine-basedβ-lactamases, and each of these classes contains significant numbers ofβ-lactamase variants, inhibition of one or a small number ofβ-lactamases is unlikely to be of therapeutic value. Legacy β-lactamaseinhibitors are largely ineffective against at least Class Acarbapenemases, against the chromosomal and plasmid-mediated Class Ccephalosporinases and against many of the Class D oxacillinases.Therefore, there is a need for improved β-lactamase inhibitorscombination therapy.

SUMMARY

Some embodiments described herein relate to a method for treating abacterial infection, comprising administering an effective amount ofCompound I or a pharmaceutically acceptable salt thereof and meropenemto a subject in need thereof:

wherein the amount of Compound I or the pharmaceutically acceptable saltthereof is from about 1.0 g to about 3.0 g and the amount of meropenemis from about 1.0 g to about 3.0 g.

Some embodiments described herein relate to a method for treating abacterial infection, comprising selecting for treatment a subject inneed for treatment of a bacterial infection who is suffering fromreduced renal function; administering an effective amount of compound Ior a pharmaceutically acceptable salt thereof and meropenem to saidsubject.

Some embodiments described herein relate to a method of treating orameliorating a lower respiratory tract infection, comprisingadministering an effective amount of Compound I or a pharmaceuticallyacceptable salt thereof and meropenem to a subject in need thereof.

In some embodiments, the method further comprises administering anadditional medicament selected from an antibacterial agent, antifungalagent, an antiviral agent, an anti-inflammatory agent, or ananti-allergic agent.

In some embodiments, the subject treated by the method described aboveis a mammal. In some further embodiments, the subject is a human.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting the plasma concentration profile (mg/L) ofvarious doses of Compound I as a function of time following a single IVinfusion in healthy subjects in the study disclosed in Example 1.

FIG. 2 is a graph depicting Compound I dose versus AUC (hr*mg/L)following single or multiple doses in healthy subjects in the studydisclosed in Example 1.

FIG. 3 is a graph depicting the plasma concentration profile (mg/L) ofCompound I alone and in combination with meropenem after 3-hourinfusions in healthy adult subjects in the study disclosed in Example 2.

FIG. 4 is a graph depicting the plasma concentration profile (mg/L) ofmeropenem alone and in combination with Compound I after 3-hourinfusions in healthy adult subjects in the study disclosed in Example 2.

FIG. 5 is a graph depicting the plasma concentration profile (mg/L) ofCompound I alone and in combination with meropenem after single and 7days of TID (i.e., three times a day) dosing by 3-hour infusions inhealthy adult subjects in the study disclosed in Example 3.

FIG. 6 is a graph depicting the plasma concentration profile (mg/L) ofmeropenem alone and in combination with Compound I after single and 7days of TID (i.e., three times a day) dosing by 3-hour infusions inhealthy adult subjects in the study disclosed in Example 3.

FIG. 7 is a graph depicting the plasma concentration profile (mg/L) of 2g Compound I alone and in combination with 2 g meropenem followingsingle and multiple doses by 3-hour infusion in healthy subjects in thestudy disclosed in Example 4.

FIG. 8 is a graph depicting the plasma concentration profile (mg/L) of 2g Compound I alone and in combination with 2 g meropenem followingsingle and multiple doses by 1-hour infusion in healthy subjects in thestudy disclosed in Example 4.

FIG. 9 is a graph depicting the mean plasma concentration profile (mg/L)of Compound I after 1-hour or 3-hour infusions of 2 g Compound I incombination with 2 g meropenem in healthy subjects in the studydisclosed in Example 4.

FIG. 10 is a graph depicting the plasma concentration profile (mg/L) of2 g meropenem alone and in combination with 2 g Compound I followingsingle and multiple doses by 3-hour infusion in healthy subjects in thestudy disclosed in Example 4.

FIG. 11 is a graph depicting the plasma concentration profile (mg/L) of2 g meropenem alone and in combination with 2 g Compound I followingsingle and multiple doses by 1-hour infusion in healthy subjects in thestudy disclosed in Example 4.

FIG. 12 is a graph depicting the mean plasma concentration profile(mg/L) of meropenem I after 1-hour or 3-hour infusions of 2 g Compound Iin combination with 2 g meropenem in healthy subjects in the studydisclosed in Example 4.

FIG. 13 is a graph depicting the plasma concentration profile (mg/L) ofmeropenem open-lactam after 1-hour infusion of 2 g meropenem alone andin combination with 2 g Compound I in healthy subjects in the studydisclosed in Example 4.

FIG. 14 is a graph depicting the mean plasma concentration profile(mg/L) of meropenem open-lactam after 1-hour or 3-hour infusions of 2 gmeropenem in combination with 2 g Compound I in healthy subjects in thestudy disclosed in Example 4.

FIG. 15 is a graph depicting the combination of 1 g Compound I and 1 gmeropenem clearance according to creatinine clearance in subjects withvarying degrees of renal impairment in the study disclosed in Example 5.

FIG. 16 is a graph depicting the mean plasma concentration profile(μg/mL) of meropenem before and after the start of the third meropenem 2g infusion over 3 hours in the study disclosed in Example 6.

FIG. 17 is a graph depicting the mean plasma concentration profile(μg/mL) of Compound I before and after the start of the third Compound I2 g infusion over 3 hours in the study disclosed in Example 6.

FIG. 18 is a graph depicting the mean plasma and epithelial lining fluid(ELF) concentration profile (μg/mL) of meropenem at time of bronchoscopywith bronchoalveolar lavage (BAL) (meropenem 2 g dose infused over 3hours) in the study disclose in Example 6.

FIG. 19 is a graph depicting the mean plasma and epithelial lining fluid(ELF) concentration profile (μg/mL) of Compound I at time ofbronchoscopy with bronchoalveolar lavage (BAL) (Compound I 2 g doseinfused over 3 hours) in the study disclosed in Example 6.

FIG. 20 is a graph depicting the mean plasma concentration profile(μg/mL) of Compound I and meropenem before and after the start of thethird meropenem 2 g/Compound I 2 g infusion over 3 hours in the studydisclosed in Example 6.

FIG. 21 is a graph depicting the mean epithelial lining fluid (ELF)concentration profile (μg/mL) of Compound I and meropenem at time ofbronchoscopy with bronchoalveolar lavage (BAL) (meropenem 2 g doseinfused over 3 hours) in the study disclosed in Example 6.

FIG. 22 is a graph depicting the activity of 1 g meropenem/1 g CompoundI administered by 3-hour infusion every 8 hours against certain strainsof Carbapenem Resistant K. pneumoniae in an in vitro Hollow Fiber Model.

FIG. 23 is a graph depicting the activity of 1 g meropenem/1 g CompoundI administered by 3-hour infusion every 8 hours against certain strainsof Carbapenem Resistant K. pneumoniae in an in vitro Hollow Fiber Model.

FIG. 24 is a graph depicting the activity of 2 g meropenem/2 g CompoundI administered by 3-hour infusion every 8 hours against certain strainsof Carbapenem Resistant K. pneumoniae in an in vitro Hollow Fiber Model.

FIG. 25 is a graph depicting the activity of 1 g meropenem/1 g CompoundI administered by 3-hour infusion every 8 hours against certain P.aeruginosa strains in an in vitro Hollow Fiber Model.

FIG. 26 is a graph depicting the representative pharmacokinetic profilesof 2 g meropenem and 2 g Compound I administered every 8 hours by 3-hourinfusion in an in vitro Hollow Fiber Model.

FIG. 27 is a graph depicting the activity of 2 g meropenem administeredevery 8 hours by 3-hour infusion against certain P. aeruginosa strainsin an in vitro Hollow Fiber Model.

FIG. 28 is a graph depicting the activity of 2 g meropenem/2 g CompoundI administered by 3-hour infusion every 8 hours against certain P.aeruginosa strains in an in vitro Hollow Fiber Model.

DETAILED DESCRIPTION OF EMBODIMENTS Definitions

The term “agent” or “test agent” includes any substance, molecule,element, compound, entity, or a combination thereof. It includes, but isnot limited to, e.g., protein, polypeptide, peptide or mimetic, smallorganic molecule, polysaccharide, polynucleotide, and the like. It canbe a natural product, a synthetic compound, or a chemical compound, or acombination of two or more substances. Unless otherwise specified, theterms “agent”, “substance”, and “compound” are used interchangeablyherein.

The term “mammal” is used in its usual biological sense. Thus, itspecifically includes humans, cattle, horses, dogs, cats, rats and micebut also includes many other species.

The term “microbial infection” refers to the invasion of the hostorganism, whether the organism is a vertebrate, invertebrate, fish,plant, bird, or mammal, by pathogenic microbes. This includes theexcessive growth of microbes that are normally present in or on the bodyof a mammal or other organism. More generally, a microbial infection canbe any situation in which the presence of a microbial population(s) isdamaging to a host mammal. Thus, a mammal is “suffering” from amicrobial infection when excessive numbers of a microbial population arepresent in or on a mammal's body, or when the effects of the presence ofa microbial population(s) is damaging the cells or other tissue of amammal. Specifically, this description applies to a bacterial infection.Note that the compounds of preferred embodiments are also useful intreating microbial growth or contamination of cell cultures or othermedia, or inanimate surfaces or objects, and nothing herein should limitthe preferred embodiments only to treatment of higher organisms, exceptwhen explicitly so specified in the claims.

The term “pharmaceutically acceptable carrier” or “pharmaceuticallyacceptable excipient” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents and the like. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions. In addition, various adjuvants such as arecommonly used in the art may be included. These and other such compoundsare described in the literature, e.g., in the Merck Index, Merck &Company, Rahway, N.J. Considerations for the inclusion of variouscomponents in pharmaceutical compositions are described, e.g., in Gilmanet al. (Eds.) (1990); Goodman and Gilman's: The Pharmacological Basis ofTherapeutics, 8th Ed., Pergamon Press.

The term “pharmaceutically acceptable salt” refers to salts that retainthe biological effectiveness and properties of the compounds of thepreferred embodiments and, which are not biologically or otherwiseundesirable. In many cases, the compounds of the preferred embodimentsare capable of forming acid and/or base salts by virtue of the presenceof amino and/or carboxyl groups or groups similar thereto.Pharmaceutically acceptable acid addition salts can be formed withinorganic acids and organic acids. Inorganic acids from which salts canbe derived include, for example, hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acidsfrom which salts can be derived include, for example, acetic acid,propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid,malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid,ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and thelike. Pharmaceutically acceptable base addition salts can be formed withinorganic and organic bases. Inorganic bases from which salts can bederived include, for example, sodium, potassium, lithium, ammonium,calcium, magnesium, iron, zinc, copper, manganese, aluminum, and thelike; particularly preferred are the ammonium, potassium, sodium,calcium and magnesium salts. Organic bases from which salts can bederived include, for example, primary, secondary, and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines, basic ion exchange resins, and the like, specificallysuch as isopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, and ethanolamine. Many such salts are known in the art,as described in WO 87/05297, Johnston et al., published Sep. 11, 1987(incorporated by reference herein in its entirety).

“Solvate” refers to the compound formed by the interaction of a solventand an EPI, a metabolite, or salt thereof. Suitable solvates arepharmaceutically acceptable solvates including hydrates.

“Subject” as used herein, means a human or a non-human mammal, e.g., adog, a cat, a mouse, a rat, a cow, a sheep, a pig, a goat, a non-humanprimate or a bird, e.g., a chicken, as well as any other vertebrate orinvertebrate.

A therapeutic effect relieves, to some extent, one or more of thesymptoms of the infection, and includes curing an infection. “Curing”means that the symptoms of active infection are eliminated, includingthe elimination of excessive members of viable microbe of those involvedin the infection. However, certain long-term or permanent effects of theinfection may exist even after a cure is obtained (such as extensivetissue damage).

“Treat,” “treatment,” or “treating,” as used herein refers toadministering a pharmaceutical composition for prophylactic and/ortherapeutic purposes. The term “prophylactic treatment” refers totreating a patient who is not yet infected, but who is susceptible to,or otherwise at risk of, a particular infection, whereby the treatmentreduces the likelihood that the patient will develop an infection. Theterm “therapeutic treatment” refers to administering treatment to apatient already suffering from an infection.

Methods of Treatment

Some embodiments described herein relate to a method for treating abacterial infection, comprising administering an effective amount ofCompound I or a pharmaceutically acceptable salt thereof and meropenemto a subject in need thereof:

wherein the amount of Compound I or the pharmaceutically acceptable saltthereof is from about 1.0 g to about 3.0 g and the amount of meropenemis from about 1.0 g to about 3.0 g.

In some embodiments, the amount of Compound I or the pharmaceuticallyacceptable salt thereof is about 2.0 g. In some embodiments, the amountof meropenem is about 2.0 g. In some embodiments, the amount of bothCompound I or the pharmaceutically acceptable salt thereof and meropenemare about 2.0 g.

In some embodiments, Compound I or the pharmaceutically acceptable saltthereof and meropenem are administered at least once per day. In someembodiments, Compound I or the pharmaceutically acceptable salt thereofand meropenem are administered 3 times per day. In some furtherembodiments, the daily dose of Compound I or the pharmaceuticallyacceptable salt thereof is about 6.0 g and wherein the daily dose ofmeropenem is about 6.0 g.

In some embodiments, the administration is by intravenous infusion. Insome such embodiments, the intravenous infusion is completed in about 1to about 5 hours. In some further embodiments, the intravenous infusionis completed is about 3 hours.

In some embodiments, Compound I or the pharmaceutically acceptable saltthereof is administered prior or subsequent to meropenem. In some otherembodiments, Compound I or the pharmaceutically acceptable salt thereofand meropenem are in a single dosage form. In some embodiments, thesingle dosage form further comprises a pharmaceutically acceptableexcipient, diluent, or carrier.

Subjects with Reduced Renal Function

Some embodiments described herein relate to a method for treating abacterial infection, comprising selecting for treatment a subject inneed for treatment of a bacterial infection who is suffering fromreduced renal function; administering an effective amount of compound Ior a pharmaceutically acceptable salt thereof and meropenem to saidsubject. In some embodiments, said subject has a creatinine clearance of≧30 ml/min and <50 ml/min. In some embodiments, said subject has acreatinine clearance of ≧20 ml/min and <30 ml/min. In some embodiments,said subject has a creatinine clearance of ≧10 ml/min and <20 ml/min. Insome embodiments, said subject has a creatinine clearance of <10 ml/min.In some embodiments, the bacterial infection is lower respiratory tractinfection.

In some embodiments, Compound I or the pharmaceutically acceptable saltthereof is administered in a dose range from about 250 mg to about 2.0g. In some further embodiments, Compound I or the pharmaceuticallyacceptable salt thereof is administered in a dose of about 500 mg toabout 1.0 g. In some such embodiments, Compound I or thepharmaceutically acceptable salt thereof is administered in a dose ofabout 1.0 g. In some other embodiments, Compound I or thepharmaceutically acceptable salt thereof is administered in a dose ofabout 500 mg. In some embodiments, meropenem is administered in a doserange from about 250 mg to about 2.0 g. In some further embodiments,meropenem is administered in a dose of about 500 mg to about 1.0 g. Insome such embodiments, meropenem is administered in a dose of about 1.0g. In some other embodiments, meropenem is administered in a dose ofabout 500 mg. In some further embodiments, both Compound I or thepharmaceutically acceptable salt thereof and meropenem are administeredin a dose of about 1.0 g. In some other embodiments, both Compound I orthe pharmaceutically acceptable salt thereof and meropenem areadministered in a dose of about 500 mg.

In some embodiments, Compound I or the pharmaceutically acceptable saltthereof and meropenem are administered at least once per day (every 24hours). In some embodiments, Compound I or the pharmaceuticallyacceptable salt thereof and meropenem are administered 2 times per day(every 12 hours). In some embodiments, Compound I or thepharmaceutically acceptable salt thereof and meropenem are administered3 times per day (every 8 hours). In some embodiments, the daily dose ofCompound I or the pharmaceutically acceptable salt thereof is about 3.0g and wherein the daily dose of meropenem is about 3.0 g. In someembodiments, the daily dose of Compound I or the pharmaceuticallyacceptable salt thereof is about 2.0 g and wherein the daily dose ofmeropenem is about 2.0 g. In some embodiments, the daily dose ofCompound I or the pharmaceutically acceptable salt thereof is about 1.0g and wherein the daily dose of meropenem is about 1.0 g. In somefurther embodiments, the daily dose of Compound I or thepharmaceutically acceptable salt thereof is about 500 mg and wherein thedaily dose of meropenem is about 500 mg.

In some embodiments, the administration is by intravenous infusion. Insome such embodiments, the intravenous infusion is completed in about 1to about 5 hours. In some further embodiments, the intravenous infusionis completed is about 3 hours.

In some embodiments, Compound I or the pharmaceutically acceptable saltthereof is administered prior or subsequent to meropenem. In some otherembodiments, Compound I or the pharmaceutically acceptable salt thereofand meropenem are in a single dosage form. In some embodiments, thesingle dosage form further comprises a pharmaceutically acceptableexcipient, diluent, or carrier.

Subjects with Lower Respiratory Tract Infection

Some embodiments described herein relate to a method of treating orameliorating a lower respiratory tract infection, comprisingadministering an effective amount of Compound I or a pharmaceuticallyacceptable salt thereof and meropenem to a subject in need thereof.

In some embodiments, Compound I or the pharmaceutically acceptable saltthereof is administered in a dose range from about 250 mg to about 5.0g. In some further embodiments, Compound I or the pharmaceuticallyacceptable salt thereof is administered in a dose range from about 1.0 gto about 3.0 g. In still some further embodiments, the amount ofCompound I is about 2.0 g. In some embodiments, meropenem isadministered in a dose range from about 250 mg to about 5.0 g. In somefurther embodiments, meropenem is administered in a dose range fromabout 1.0 g to about 3.0 g. In still some further embodiments, theamount of meropenem is about 2.0 g. In some embodiments, both Compound Ior the pharmaceutically acceptable salt thereof and meropenem areadministered in a dose of about 2.0 g.

In some embodiments, Compound I or the pharmaceutically acceptable saltthereof and meropenem are administered at least once per day. In someembodiments, Compound I or the pharmaceutically acceptable salt thereofand meropenem are administered 3 times per day. In some embodiments, thedaily dose of Compound I or the pharmaceutically acceptable salt thereofis from about 3.0 g to about 6.0 g and wherein the daily dose ofmeropenem is from about 3.0 g to about 6.0 g. In some furtherembodiments, the daily dose of Compound I or the pharmaceuticallyacceptable salt thereof is about 6.0 g and wherein the daily dose ofmeropenem is about 6.0 g.

In some embodiments, the administration is by intravenous infusion. Insome such embodiments, the intravenous infusion is completed in about 1to about 5 hours. In some further embodiments, the intravenous infusionis completed is about 3 hours.

In some embodiments, Compound I or the pharmaceutically acceptable saltthereof is administered prior or subsequent to meropenem. In some otherembodiments, Compound I or the pharmaceutically acceptable salt thereofand meropenem are in a single dosage form. In some embodiments, thesingle dosage form further comprises a pharmaceutically acceptableexcipient, diluent, or carrier.

In any embodiments of the methods described herein, the method mayfurther comprise administering an additional medicament selected from anantibacterial agent, antifungal agent, an antiviral agent, ananti-inflammatory agent, or an anti-allergic agent.

In some embodiments, the subject treated by the method described aboveis a mammal. In some further embodiments, the subject is a human.

In any embodiments of the methods described herein, the treatment is forinfection caused by carbapenem-resistant Enterobacteriaceae.

Indications

The compositions comprising Compound I and a carbapenem compoundmeropenem described herein can be used to treat bacterial infections.Bacterial infections that can be treated with a combination of CompoundI and meropenem can comprise a wide spectrum of bacteria. Exampleorganisms include gram-positive bacteria, gram-negative bacteria,aerobic and anaerobic bacteria, such as Staphylococcus, Lactobacillus,Streptococcus, Sarcina, Escherichia, Enterobacter, Klebsiella,Pseudomonas, Acinetobacter, Mycobacterium, Proteus, Campylobacter,Citrobacter, Nisseria, Baccillus, Bacteroides, Peptococcus, Clostridium,Salmonella, Shigella, Serratia, Haemophilus, Brucella and otherorganisms.

More examples of bacterial infections include Pseudomonas aeruginosa,Pseudomonas fluorescens, Pseudomonas acidovorans, Pseudomonasalcaligenes, Pseudomonas putida, Stenotrophomonas maltophilia,Burkholderia cepacia, Aeromonas hydrophilia, Escherichia coli,Citrobacter freundii, Salmonella typhimurium, Salmonella typhi,Salmonella paratyphi, Salmonella enteritidis, Shigella dysenteriae,Shigella flexneri, Shigella sonnei, Enterobacter cloacae, Enterobacteraerogenes, Klebsiella pneumoniae, Klebsiella oxytoca, Serratiamarcescens, Francisella tularensis, Morganella morganii, Proteusmirabilis, Proteus vulgaris, Providencia alcalifaciens, Providenciarettgeri, Providencia stuartii, Acinetobacter baumannii, Acinetobactercalcoaceticus, Acinetobacter haemolyticus, Yersinia enterocolitica,Yersinia pestis, Yersinia pseudotuberculosis, Yersinia intermedia,Bordetella pertussis, Bordetella parapertussis, Bordetellabronchiseptica, Haemophilus influenzae, Haemophilus parainfluenzae,Haemophilus haemolyticus, Haemophilus parahaemolyticus, Haemophilusducreyi, Pasteurella multocida, Pasteurella haemolytica, Branhamellacatarrhalis, Helicobacter pylori, Campylobacter fetus, Campylobacterjejuni, Campylobacter coli, Borrelia burgdorferi, Vibrio cholerae,Vibrio parahaemolyticus, Legionella pneumophila, Listeria monocytogenes,Neisseria gonorrhoeae, Neisseria meningitidis, Kingella, Moraxella,Gardnerella vaginalis, Bacteroides fragilis, Bacteroides distasonis,Bacteroides 3452A homology group, Bacteroides vulgatus, Bacteroidesovalus, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroideseggerthii, Bacteroides splanchnicus, Clostridium difficile,Mycobacterium tuberculosis, Mycobacterium avium, Mycobacteriumintracellulare, Mycobacterium leprae, Corynebacterium diphtheriae,Corynebacterium ulcerans, Streptococcus pneumoniae, Streptococcusagalactiae, Streptococcus pyogenes, Enterococcus faecalis, Enterococcusfaecium, Staphylococcus aureus, Staphylococcus epidermidis,Staphylococcus saprophyticus, Staphylococcus intermedius, Staphylococcushyicus subsp. hyicus, Staphylococcus haemolyticus, Staphylococcushominis, or Staphylococcus saccharolyticus.

In some embodiments, the infection is caused by a bacteria selected fromPseudomonas aeruginosa, Pseudomonas fluorescens, Stenotrophomonasmaltophilia, Escherichia coli, Citrobacter freundii, Salmonellatyphimurium, Salmonella typhi, Salmonella paratyphi, Salmonellaenteritidis, Shigella dysenteriae, Shigella flexneri, Shigella sonnei,Enterobacter cloacae, Enterobacter aerogenes, Klebsiella pneumoniae,Klebsiella oxytoca, Serratia marcescens, Acinetobacter calcoaceticus,Acinetobacter haemolyticus, Yersinia enterocolitica, Yersinia pestis,Yersinia pseudotuberculosis, Yersinia intermedia, Haemophilusinfluenzae, Haemophilus parainfluenzae, Haemophilus haemolyticus,Haemophilus parahaemolyticus, Helicobacter pylori, Campylobacter fetus,Campylobacter jejuni, Campylobacter coli, Vibrio cholerae, Vibrioparahaemolyticus, Legionella pneumophila, Listeria monocytogenes,Neisseria gonorrhoeae, Neisseria meningitidis, Moraxella, Bacteroidesfragilis, Bacteroides vulgatus, Bacteroides ovalus, Bacteroidesthetaiotaomicron, Bacteroides uniformis, Bacteroides eggerthii, orBacteroides splanchnicus.

In some embodiments, the bacterial infection is gram-negative infection.In some embodiments, the bacterial infection is lower respiratory tractinfection. In some embodiments, the bacterial infection is caused byPseudomonas aeruginosa. In some embodiments, the bacterial infection iscaused by Klebsiella pneumonia.

Antibacterial Compounds

Compound I has the structures shown as follows:

In some embodiments, due to the facile exchange of boron esters,Compound I may convert to or exist in equilibrium with alternate forms.Accordingly, in some embodiments, Compound I may exist in combinationwith one or more of these forms. For example, Compound I may exist incombination with one or more open-chain form (Formula Ia), dimeric form(Formula Ib), cyclic dimeric form (Formula Ic), trimeric form (FormulaId), cyclic trimeric form (Formula Ie), and the like. Compound I and itsenantiomer, diastereoisomer or tautomer, or pharmaceutically acceptablesalt is described in U.S. Pat. No. 8,680,136, which is incorporated byreference in its entirety.

Meropenem is an ultra-broad-spectrum injectable antibiotic used to treata wide variety of infections. It is a β-lactam and belongs to thesubgroup of carbapenem. It has the structure shown as follows:

Some embodiments include methods for treating or preventing a bacterialinfection comprising administering to a subject in need thereof, aneffective amount of Compound I and meropenem, wherein Compound I can bein any one of the forms described above or a combination thereof.

Some embodiments further comprise administering an additionalmedicament, either is a separate composition or in the same composition.In some embodiments, the additional medicament includes an antibacterialagent, antifungal agent, an antiviral agent, an anti-inflammatory agentor an anti-allergic agent. In some embodiments, the additionalmedicament comprises an antibacterial agent such as an additionalβ-lactam.

In some embodiments, the additional β-lactam includes Amoxicillin,Ampicillin (Pivampicillin, Hetacillin, Bacampicillin, Metampicillin,Talampicillin), Epicillin, Carbenicillin (Carindacillin), Ticarcillin,Temocillin, Azlocillin, Piperacillin, Mezlocillin, Mecillinam(Pivmecillinam), Sulbenicillin, Benzylpenicillin (G), Clometocillin,Benzathine benzylpenicillin, Procaine benzylpenicillin, Azidocillin,Penamecillin, Phenoxymethylpenicillin (V), Propicillin, Benzathinephenoxymethylpenicillin, Pheneticillin, Cloxacillin (Dicloxacillin,Flucloxacillin), Oxacillin, Meticillin, Nafcillin, Faropenem, Biapenem,Doripenem, Ertapenem, Imipenem, Panipenem, Tomopenem, Razupenem,Tebipenem, Sulopenem, Cefazolin, Cefacetrile, Cefadroxil, Cefalexin,Cefaloglycin, Cefalonium, Cefaloridine, Cefalotin, Cefapirin,Cefatrizine, Cefazedone, Cefazaflur, Cefradine, Cefroxadine, Ceftezole,Cefaclor, Cefamandole, Cefminox, Cefonicid, Ceforanide, Cefotiam,Cefprozil, Cefbuperazone, Cefuroxime, Cefuzonam, Cefoxitin, Cefotetan,Cefmetazole, Loracarbef, Cefixime, Ceftazidime, Ceftriaxone, Cefcapene,Cefdaloxime, Cefdinir, Cefditoren, Cefetamet, Cefmenoxime, Cefodizime,Cefoperazone, Cefotaxime, Cefpimizole, Cefpiramide, Cefpodoxime,Cefsulodin, Cefteram, Ceftibuten, Ceftiolene, Ceftizoxime, Flomoxef,Latamoxef, Cefepime, Cefozopran, Cefpirome, Cefquinome, Ceftobiprole,Ceftaroline, Ceftolozane, CXA-101, RWJ-54428, MC-04,546, ME1036,BAL30072, SYN 2416, Ceftiofur, Cefquinome, Cefovecin, Aztreonam,Tigemonam, Carumonam, RWJ-442831, RWJ-333441, RWJ-333442, S649266,GSK3342830, and AIC 499.

In some embodiments, the additional β-lactam includes Ceftazidime,Doripenem, Ertapenem, Imipenem, or Panipenem.

Some embodiments include a pharmaceutical composition comprising atherapeutically effective amount of any one of the foregoing compoundsand a pharmaceutically acceptable excipient.

Administration and Pharmaceutical Compositions

Some embodiments include pharmaceutical compositions comprising: (a) asafe and therapeutically effective amount of compound I, or itscorresponding enantiomer, diastereoisomer or tautomer, orpharmaceutically acceptable salt; (b) meropenem, and (c) apharmaceutically acceptable carrier.

Compound I and meropenem are administered at a therapeutically effectivedosage, e.g., a dosage sufficient to provide treatment for the diseasestates previously described. In some embodiments, a single dose ofCompound I and meropenem may range from about 250 mg to about 5000 mg orfrom about 1000 mg to about 3000 mg. In some embodiments, Compound I andmeropenem can be administered at least once a day, for example 1 to 5times a day.

Administration of the combination comprising Compound I or itscorresponding enantiomer, diastereoisomer, tautomer, or thepharmaceutically acceptable salt thereof and meropenem can be via any ofthe accepted modes of administration for agents that serve similarutilities including, but not limited to, orally, subcutaneously,intravenously, intranasally, topically, transdermally,intraperitoneally, intramuscularly, intrapulmonarilly, vaginally,rectally, or intraocularly. Intravenous, oral and parenteraladministrations are customary in treating the indications that are thesubject of the preferred embodiments.

Compound I and meropenem can be formulated into pharmaceuticalcompositions for use in treatment of these conditions. Standardpharmaceutical formulation techniques are used, such as those disclosedin Remington's The Science and Practice of Pharmacy, 21st Ed.,Lippincott Williams & Wilkins (2005), incorporated by reference in itsentirety.

In addition to Compound I and meropenem, some embodiments includecompositions containing a pharmaceutically-acceptable carrier. The term“pharmaceutically-acceptable carrier”, as used herein, means one or morecompatible solid or liquid filler diluents or encapsulating substances,which are suitable for administration to a mammal. The term“compatible”, as used herein, means that the components of thecomposition are capable of being commingled with the subject compound,and with each other, in a manner such that there is no interaction,which would substantially reduce the pharmaceutical efficacy of thecomposition under ordinary use situations. Pharmaceutically-acceptablecarriers must, of course, be of sufficiently high purity andsufficiently low toxicity to render them suitable for administrationpreferably to an animal, preferably mammal being treated.

Some examples of substances, which can serve aspharmaceutically-acceptable carriers or components thereof, are sugars,such as lactose, glucose and sucrose; starches, such as corn starch andpotato starch; cellulose and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose, and methyl cellulose; powderedtragacanth; malt; gelatin; talc; solid lubricants, such as stearic acidand magnesium stearate; calcium sulfate; vegetable oils, such as peanutoil, cottonseed oil, sesame oil, olive oil, corn oil and oil oftheobroma; polyols such as propylene glycol, glycerine, sorbitol,mannitol, and polyethylene glycol; alginic acid; emulsifiers, such asthe TWEENS; wetting agents, such sodium lauryl sulfate; coloring agents;flavoring agents; tableting agents, stabilizers; antioxidants;preservatives; pyrogen-free water; isotonic saline; and phosphate buffersolutions.

The choice of a pharmaceutically-acceptable carrier to be used inconjunction with the combination is basically determined by the way thecombination is to be administered.

The compositions described herein are preferably provided in unit dosageform. As used herein, a “unit dosage form” is a composition containingan amount of a compound that is suitable for administration to ananimal, preferably mammal subject, in a single dose, according to goodmedical practice. The preparation of a single or unit dosage formhowever, does not imply that the dosage form is administered once perday or once per course of therapy. Such dosage forms are contemplated tobe administered once, twice, thrice or more per day and may beadministered as infusion over a period of time (e.g., from about 30minutes to about 2-6 hours), or administered as a continuous infusion,and may be given more than once during a course of therapy, though asingle administration is not specifically excluded. The skilled artisanwill recognize that the formulation does not specifically contemplatethe entire course of therapy and such decisions are left for thoseskilled in the art of treatment rather than formulation.

The compositions useful as described above may be in any of a variety ofsuitable forms for a variety of routes for administration, for example,for oral, nasal, rectal, topical (including transdermal), ocular,intracerebral, intracranial, intrathecal, intra-arterial, intravenous,intramuscular, or other parental routes of administration. The skilledartisan will appreciate that oral and nasal compositions comprisecompositions that are administered by inhalation, and made usingavailable methodologies. Depending upon the particular route ofadministration desired, a variety of pharmaceutically-acceptablecarriers well-known in the art may be used. Pharmaceutically-acceptablecarriers include, for example, solid or liquid fillers, diluents,hydrotropies, surface-active agents, and encapsulating substances.Optional pharmaceutically-active materials may be included, which do notsubstantially interfere with the inhibitory activity of the compound.The amount of carrier employed in conjunction with the compound issufficient to provide a practical quantity of material foradministration per unit dose of the compound. Techniques andcompositions for making dosage forms useful in the methods describedherein are described in the following references, all incorporated byreference herein: Modern Pharmaceutics, 4th Ed., Chapters 9 and 10(Banker & Rhodes, editors, 2002); Lieberman et al., PharmaceuticalDosage Forms: Tablets (1989); and Ansel, Introduction to PharmaceuticalDosage Forms 8th Edition (2004). In some embodiments, the pharmaceuticalcompositions are administered intravenously. In some embodiments, thepharmaceutical compositions are administered orally. In some otherembodiments, the pharmaceutical compositions are administeredintraperitoneally.

Various oral dosage forms can be used, including such solid forms astablets, capsules, granules and bulk powders. These oral forms comprisea safe and effective amount, usually at least about 5%, with a maximumof about 90%, of the compound. Tablets can be compressed, tablettriturates, enteric-coated, sugar-coated, film-coated, ormultiple-compressed, containing suitable binders, lubricants, diluents,disintegrating agents, coloring agents, flavoring agents, flow-inducingagents, and melting agents. Liquid oral dosage forms include aqueoussolutions, emulsions, suspensions, solutions and/or suspensionsreconstituted from non-effervescent granules, and effervescentpreparations reconstituted from effervescent granules, containingsuitable solvents, preservatives, emulsifying agents, suspending agents,diluents, sweeteners, melting agents, coloring agents and flavoringagents.

The pharmaceutically-acceptable carrier suitable for the preparation ofunit dosage forms for peroral administration is well-known in the art.Tablets typically comprise conventional pharmaceutically-compatibleadjuvants as inert diluents, such as calcium carbonate, sodiumcarbonate, mannitol, lactose and cellulose; binders such as starch,gelatin and sucrose; disintegrants such as starch, alginic acid andcroscarmelose; lubricants such as magnesium stearate, stearic acid andtalc. Glidants such as silicon dioxide can be used to improve flowcharacteristics of the powder mixture. Coloring agents, such as the FD&Cdyes, can be added for appearance. Sweeteners and flavoring agents, suchas aspartame, saccharin, menthol, peppermint, and fruit flavors, areuseful adjuvants for chewable tablets. Capsules typically comprise oneor more solid diluents disclosed above. The selection of carriercomponents depends on secondary considerations like taste, cost, andshelf stability, which are not critical, and can be readily made by aperson skilled in the art.

Peroral compositions also include liquid solutions, emulsions,suspensions, and the like. The pharmaceutically-acceptable carrierssuitable for preparation of such compositions are well known in the art.Typical components of carriers for syrups, elixirs, emulsions andsuspensions include ethanol, glycerol, propylene glycol, polyethyleneglycol, liquid sucrose, sorbitol and water. For a suspension, typicalsuspending agents include methyl cellulose, sodium carboxymethylcellulose, AVICEL RC-591, tragacanth and sodium alginate; typicalwetting agents include lecithin and polysorbate 80; and typicalpreservatives include methyl paraben and sodium benzoate. Peroral liquidcompositions may also contain one or more components such as sweeteners,flavoring agents and colorants disclosed above.

Such compositions may also be coated by conventional methods, typicallywith pH or time-dependent coatings, such that the subject compound isreleased in the gastrointestinal tract in the vicinity of the desiredtopical application, or at various times to extend the desired action.Such dosage forms typically include, but are not limited to, one or moreof cellulose acetate phthalate, polyvinylacetate phthalate,hydroxypropyl methyl cellulose phthalate, ethyl cellulose, Eudragitcoatings, waxes and shellac.

Compositions described herein may optionally include other drug actives.

Other compositions useful for attaining systemic delivery of the subjectcompounds include sublingual, buccal and nasal dosage forms. Suchcompositions typically comprise one or more of soluble filler substancessuch as sucrose, sorbitol and mannitol; and binders such as acacia,microcrystalline cellulose, carboxymethyl cellulose and hydroxypropylmethyl cellulose. Glidants, lubricants, sweeteners, colorants,antioxidants and flavoring agents disclosed above may also be included.

A liquid composition, which is formulated for topical ophthalmic use, isformulated such that it can be administered topically to the eye. Thecomfort should be maximized as much as possible, although sometimesformulation considerations (e.g. drug stability) may necessitate lessthan optimal comfort. In the case that comfort cannot be maximized, theliquid should be formulated such that the liquid is tolerable to thepatient for topical ophthalmic use. Additionally, an ophthalmicallyacceptable liquid should either be packaged for single use, or contain apreservative to prevent contamination over multiple uses.

For ophthalmic application, solutions or medicaments are often preparedusing a physiological saline solution as a major vehicle. Ophthalmicsolutions should preferably be maintained at a comfortable pH with anappropriate buffer system. The formulations may also containconventional, pharmaceutically acceptable preservatives, stabilizers andsurfactants.

Preservatives that may be used in the pharmaceutical compositionsdisclosed herein include, but are not limited to, benzalkonium chloride,PHMB, chlorobutanol, thimerosal, phenylmercuric, acetate andphenylmercuric nitrate. A useful surfactant is, for example, Tween 80.Likewise, various useful vehicles may be used in the ophthalmicpreparations disclosed herein. These vehicles include, but are notlimited to, polyvinyl alcohol, povidone, hydroxypropyl methyl cellulose,poloxamers, carboxymethyl cellulose, hydroxyethyl cellulose and purifiedwater.

Tonicity adjustors may be added as needed or convenient. They include,but are not limited to, salts, particularly sodium chloride, potassiumchloride, mannitol and glycerin, or any other suitable ophthalmicallyacceptable tonicity adjustor.

Various buffers and means for adjusting pH may be used so long as theresulting preparation is ophthalmically acceptable. For manycompositions, the pH will be between 4 and 9. Accordingly, buffersinclude acetate buffers, citrate buffers, phosphate buffers and boratebuffers. Acids or bases may be used to adjust the pH of theseformulations as needed.

In a similar vein, an ophthalmically acceptable antioxidant includes,but is not limited to, sodium metabisulfite, sodium thiosulfate,acetylcysteine, butylated hydroxyanisole and butylated hydroxytoluene.

Other excipient components, which may be included in the ophthalmicpreparations, are chelating agents. A useful chelating agent is edetatedisodium, although other chelating agents may also be used in place orin conjunction with it.

For topical use, creams, ointments, gels, solutions or suspensions,etc., containing the compound disclosed herein are employed. Topicalformulations may generally be comprised of a pharmaceutical carrier,co-solvent, emulsifier, penetration enhancer, preservative system, andemollient.

For intravenous administration, the compounds and compositions describedherein may be dissolved or dispersed in a pharmaceutically acceptablediluent, such as a saline or dextrose solution. Suitable excipients maybe included to achieve the desired pH, including but not limited toNaOH, sodium carbonate, sodium acetate, HCl, and citric acid. In variousembodiments, the pH of the final composition ranges from 2 to 8, orpreferably from 4 to 7. Antioxidant excipients may include sodiumbisulfite, acetone sodium bisulfite, sodium formaldehyde, sulfoxylate,thiourea, and EDTA. Other non-limiting examples of suitable excipientsfound in the final intravenous composition may include sodium orpotassium phosphates, citric acid, tartaric acid, gelatin, andcarbohydrates such as dextrose, mannitol, and dextran. Furtheracceptable excipients are described in Powell, et al., Compendium ofExcipients for Parenteral Formulations, PDA J Pharm Sci and Tech 1998,52 238-311 and Nema et al., Excipients and Their Role in ApprovedInjectable Products: Current Usage and Future Directions, PDA J PharmSci and Tech 2011, 65 287-332, both of which are incorporated herein byreference in their entirety. Antimicrobial agents may also be includedto achieve a bacteriostatic or fungistatic solution, including but notlimited to phenylmercuric nitrate, thimerosal, benzethonium chloride,benzalkonium chloride, phenol, cresol, and chlorobutanol.

The resulting composition may be infused into the patient over a periodof time. In various embodiments, the infusion time ranges from 5 minutesto continuous infusion, from 10 minutes to 8 hours, from 30 minutes to 4hours, and from 1 hour to 3 hours. In one embodiment, the drug isinfused over a 3 hour period. The infusion may be repeated at thedesired dose interval, which may include, for example, 6 hours, 8 hours,12 hours, or 24 hours.

The compositions for intravenous administration may be provided tocaregivers in the form of one more solids that are reconstituted with asuitable diluent such as sterile water, saline or dextrose in watershortly prior to administration. Reconstituted concentrated solutionsmay be further diluted into a parenteral solutions having a volume offrom about 25 to about 1000 ml, from about 30 ml to about 500 ml, orfrom about 50 ml to about 250 ml. In other embodiments, the compositionsare provided in solution ready to administer parenterally. In stillother embodiments, the compositions are provided in a solution that isfurther diluted prior to administration. In embodiments that includeadministering a combination of a compound described herein and anotheragent, the combination may be provided to caregivers as a mixture, orthe caregivers may mix the two agents prior to administration, or thetwo agents may be administered separately.

The actual dose of the active compounds described herein depends on thespecific compound, and on the condition to be treated; the selection ofthe appropriate dose is well within the knowledge of the skilledartisan.

Kits for Intravenous Administration

Some embodiments include a kit comprising Compound I and a carbapenemantibacterial agent Meropenem. In some embodiments, the kits are usedfor intravenous administration.

In one embodiment, both components are provided in a single sterilecontainer. In the case of solids for reconstitution, the agents may bepre-blended and added to the container simultaneously or may bedry-powder filled into the container in two separate steps. In someembodiments, the solids are sterile crystalline products. In otherembodiment, the solids are lyophiles. In one embodiment, both componentsare lyophilized together. Non-limiting examples of agents to aid inlyophilization include sodium or potassium phosphates, citric acid,tartaric acid, gelatin, and carbohydrates such as dextrose, mannitol,and dextran. One embodiment includes non-sterile solids that areirradiated either before or after introduction into the container.

In the case of a liquid, the agents may be dissolved or dispersed in adiluent ready for administration. In another embodiment, the solution ordispersion may be further diluted prior to administration. Someembodiments include providing the liquid in an IV bag. The liquid may befrozen to improve stability.

In one embodiment, the container includes other ingredients such as a pHadjuster, a solubilizing agent, or a dispersing agent. Non-limitingexamples of pH adjusters include NaOH, sodium carbonate, sodium acetate,HCl, and citric acid.

In an alternative embodiment, the two components may be provided inseparate containers. Each container may include a solid, solution, ordispersion. In such embodiments, the two containers may be provided in asingle package or may be provided separately. In one embodiment, thecompound described herein is provided as a solution while the additionalagent (e.g., antibacterial agent) is provided as a solid ready forreconstitution. In one such embodiment, the solution of the compounddescribed herein is used as the diluent to reconstitute the other agent.

In some embodiments, the kit may comprise comprises one or moreadditional medicaments selected from an antibacterial agent, antifungalagent, an antiviral agent, an anti-inflammatory agent, or ananti-allergic agent. The additional medicaments can be prepared in thesame way as described above.

EXAMPLES

The following examples, including experiments and results achieved, areprovided for illustrative purposes only and are not to be construed aslimiting the present application.

Example 1

Example 1 provides a summary of a clinical study of the safety,tolerability and pharmacokinetics of the beta-lactamase inhibitorCompound I in healthy adult subjects.

Methods:

56 healthy subjects were enrolled into one of 7 cohorts of 8 subjectseach in the single ascending dose phase (250 mg, 500 mg, 750 mg, 1000mg, 1250 mg, 1500 mg and 2000 mg). Thirty-two additional subjects werethen enrolled into one of 4 cohorts in the multiple-dose phase (250 mg,1000 mg, 1500 mg, and 2000 mg, given q8h for 7 days). Within eachcohort, subjects were randomly assigned to Compound I (n=6) or normalsaline placebo (n=2). All infusions were administered over 3 hours.Plasma and urine samples were obtained after single or multiple dosesand assayed for Compound I content using a validated HPLC/MS method.

Results:

Table 1 summarizes mean pharmacokinetics of Compound I in differentdoses. Compound I concentration profile as a function of time followinga single IV fusion and Compound I AUC profile as a function of dose areillustrated in FIGS. 1 and 2 respectively.

TABLE 1 Compound I Dose, mg Parameters 250 250 500 750 1000 1000 (Mean ±SD) SD MD SD SD SD MD C_(max), μg/mL 5.03 ± 0.86  4.81 ± 1.04  9.97 ±0.95 15.30 ± 2.76 21.80 ± 3.83 21.30 ± 6.63 T_(max), h 3.02 2.25 3 33.01 3 T_(1/2), h 1.17 ± 0.15  1.17 ± 0.13  1.35 ± 0.22  1.24 ± 0.33 1.41 ± 0.28  1.43 ± 0.36 AUC_((0-t)), μg · h/mL 16.2 ± 3.17 16.30 ±3.56 34.50 ± 4.87 50.60 ± 7.51  76.70 ± 13.20  74.60 ± 17.90AUC_((0-inf)), μg · h/mL 16.6 ± 3.24 35.60 ± 5.21 51.80 ± 8.03  79.30 ±14.20 CL, L/h 15.6 ± 2.63 15.20 ± 2.56 14.30 ± 2.28 14.80 ± 2.24 13.10 ±2.59 14.10 ± 3.42 V_(ss), L 24.5 ± 5.81 25.40 ± 2.96 23.20 ± 3.97 21.00± 3.03 V_(d), L 26.2 ± 5.30 25.70 ± 5.57 27.70 ± 4.64 23.20 ± 3.97 25.90± 3.80 28.00 ± 5.66 CL, L/h/kg 0.20 ± 0.03  0.20 ± 0.02  0.19 ± 0.02 0.19 ± 0.04  0.17 ± 0.02  0.18 ± 0.02 V_(ss), L/kg 0.31 ± 0.06  0.33 ±0.04  0.30 ± 0.09  0.28 ± 0.02 V_(d), L/kg 0.33 ± 0.06  0.34 ± 0.06 0.37 ± 0.07  0.34 ± 0.10  0.33 ± 0.06  0.36 ± 0.05 CL_(R), L/h 12.70 ±2.71  12.70 ± 3.68 11.80 ± 1.63 13.00 ± 2.08 12.10 ± 2.43 11.70 ± 3.75Urinary Recovery % 81.30 ± 16.60  79.90 ± 16.30 80.30 ± 9.94 86.40 ±5.05 89.90 ± 6.97  82.80 ± 10.30 CL_(Non-R), L/h 2.85 ± 3.11  2.49 ±3.27  2.55 ± 1.92  1.72 ± 0.80  0.97 ± 0.92  2.31 ± 1.21 Compound IDose, mg Parameters 1250 1500 1500 2000 2000 (Mean ± SD) SD SD MD SD MDC_(max), μg/mL 27.80 ± 3.67 32.90 ± 5.77 33.40 ± 4.48 41.60 ± 4.75 40.90± 4.68 T_(max), h 3 3.01 3 3.02 2.25 T_(1/2), h  1.32 ± 0.47  1.40 ±0.31  1.65 ± 0.26  1.51 ± 0.08  1.66 ± 0.10 AUC_((0-t)), μg · h/mL 97.20 ± 14.80 110.00 ± 18.90 118.00 ± 15.30 140.00 ± 13.50 145.00 ±15.80 AUC_((0-inf)), μg · h/mL 100.00 ± 17.40 114.00 ± 20.00 144.00 ±13.90 CL, L/h 12.80 ± 2.36 13.50 ± 2.17 12.90 ± 1.71 14.00 ± 1.40 14.00± 1.78 V_(ss), L 20.20 ± 2.43 23.00 ± 4.76 21.80 ± 2.26 V_(d), L 20.20 ±2.43 26.90 ± 5.39 30.30 ± 3.48 30.60 ± 4.45 33.40 ± 4.52 CL, L/h/kg 0.18 ± 0.04  0.16 ± 0.02  0.15 ± 0.01  0.17 ± 0.03  0.17 ± 0.02 V_(ss),L/kg  0.29 ± 0.05  0.28 ± 0.05  0.27 ± 0.04 V_(d), L/kg  0.33 ± 0.10 0.33 ± 0.07  0.37 ± 0.07  0.38 ± 0.07  0.41 ± 0.05 CL_(R), L/h 11.50 ±2.58 11.80 ± 1.88 11.20 ± 1.72 15.10 ± 2.55 12.80 ± 2.05 UrinaryRecovery % 86.90 ± 9.71 86.60 ± 7.22 86.80 ± 2.48 105.00 ± 15.10 91.60 ±5.36 CL_(Non-R), L/h  1.33 ± 1.31  1.42 ± 1.17  1.68 ± 0.13 −1.07 ± 2.13 1.15 ± 0.68

Maximum concentrations for Compound I were achieved at the end of the3-hour infusion. Compound I exposure (Cmax and AUC) increased in adose-proportional manner following single and multiple doses (See FIGS.1 and 2). There was no evidence of accumulation with multiple doses,consistent with the observed terminal half-life (<2 hours). Both thevolume of distribution and plasma clearance were independent of dose.High concentrations of FIGS. 1 and 2 were measured in the urine. Urinaryrecovery was 80% or greater over 48 hours across all dose groups.

No subjects discontinued the study due to adverse events (AEs) and noserious adverse events (SAEs) were observed. AEs were similar betweenCompound I and placebo-treated subjects, with no evidence of increasingincidence or severity of AEs with increasing dose, and all AEs were mildor moderate.

Conclusion:

Compound I was safe and well tolerated at all doses tested. AUC and Cmaxincreased proportionally independent of dose.

Example 2

Example 2 provides a summary of a clinical study of the safety,tolerability and pharmacokinetics of the beta-lactamase inhibitorCompound I alone, meropenem alone, and the combination of both inhealthy adult subjects.

Methods:

Eighty healthy subjects were enrolled into 1 of 5 cohorts in the singleascending dose phase (250 mg, 1000 mg, 1500 mg and 2000 mg Compound I incombination with 1 or 2 g of meropenem). Within each cohort, subjectswere administered single doses of either Compound I or meropenem day 1,and Compound I or meropenem day 3. The combination of both drugs wasadministered on day 7. All drugs were infused over 3 hours. Plasma andurine samples were obtained and assayed using validated HPLC/MS methods.Pharmacokinetics of Compound I alone and in combination with meropenemafter 3-hour infusions in healthy adult subjects and pharmacokinetics ofmeropenem alone and in combination with Compound I after 3-hourinfusions in healthy adult subjects are illustrated in FIGS. 3 and 4respectively.

Results:

Pharmacokinetic parameters, derived using non-compartmental methods, foreach drug alone and in combination of Compound I and meropenem are shownbelow in Table 2 and Table 3. Table 2 summarizes Compound Ipharmacokinetic parameters following single dose of Compound Iadministered alone or in combination with meropenem as 3-hour infusionsto healthy volunteers (data are mean±standard deviation). Table 3summarizes meropenem pharmacokinetic parameters following single dose ofmeropenem administered alone or in combination with Compound I as 3-hourinfusions to healthy volunteers (data are mean±standard deviation).

TABLE 2 Compound I 250 mg Compound I 1000 mg Compound I 1500 mgMeropenem Meropenem Meropenem Alone 1 g Alone 1 g Alone 1 g Parameter (N= 24) (N = 8) (N = 5) (N = 5) (N = 8) (N = 7) C_(max) (mg/L)  5.20 ±0.92  5.34 ± 0.78 21.98 ± 3.54 23.68 ± 4.38 37.23 ± 5.33 37.14 ± 4.70AUC_((0-∞)) (mg · h/L) 17.48 ± 3.02 17.40 ± 2.22  77.56 ± 15.87  81.18 ±15.38 123.66 ± 18.03 127.07 ± 20.99 Half-Life (h)  1.18 ± 0.35  1.08 ±0.21  1.56 ± 0.67  1.53 ± 0.32  1.21 ± 0.24  1.35 ± 0.22 Vss (L) 23.15 ±6.00 22.25 ± 3.02 21.44 ± 5.22 20.25 ± 3.20 19.37 ± 5.14 19.83 ± 2.84Plasma Clearance 14.69 ± 2.38 14.56 ± 1.76 13.35 ± 2.83 12.70 ± 2.5012.35 ± 1.75 12.04 ± 1.70 (L/h) Compound I 2000 mg Compound I 2000 mgMeropenem Meropenem Alone 1 g Alone 2 g Parameter (N = 8) (N = 8) (N =8) (N = 8) C_(max) (mg/L) 39.20 ± 4.29 41.44 ± 4.38  51.44 ± 16.16 51.66± 7.26 AUC_((0-∞)) (mg · h/L) 133.26 ± 20.89 141.02 ± 21.35 159.21 ±44.58 170.44 ± 31.99 Half-Life (h)  1.31 ± 0.32  1.43 ± 0.22  1.39 ±0.20  1.98 ± 0.81 Vss (L) 22.02 ± 2.24 22.43 ± 2.00 21.37 ± 3.33 21.84 ±3.50 Plasma Clearance 15.32 ± 2.33 14.44 ± 1.97 13.43 ± 3.23 12.08 ±2.09 (L/h) C_(max) = maximum observed drug concentration; AUC(0-Tlast) =area under the drug concentration-time curve from time zero to time tlast; Vss = apparent volume of distribution at steady state

TABLE 3 Meropenem 1 g Compound I Compound I Alone 250 mg Alone 1000 mgAlone Parameter (N = 24) (N = 8) (N = 9) (N = 5) (N = 13) C_(max) (mg/L)16.35 ± 3.04 17.17 ± 4.81 18.93 ± 3.65 20.16 ± 3.97 20.75 ± 2.23AUC_((0-∞)) (mg · h/L) 51.32 ± 8.88  52.31 ± 12.88  59.77 ± 12.09  65.88± 15.33 64.97 ± 8.86 Half-Life (h)  0.98 ± 0.18  0.91 ± 0.14  0.96 ±0.11  1.15 ± 0.21  0.89 ± 0.08 Vss (L) 25.86 ± 6.55 22.18 ± 2.63 21.59 ±3.21 21.06 ± 4.50 18.89 ± 2.62 Plasma Clearance 20.04 ± 3.40 16.94 ±2.47 17.39 ± 3.71 15.84 ± 3.57 15.64 ± 1.98 (L/h) Meropenem 1 gMeropenem 2 g Compound I Compound I Compound I 1500 mg Alone 2000 mgAlone 2000 mg Parameter (N = 7) (N = 14) (N = 7) (N = 14) (N = 8)C_(max) (mg/L) 20.76 ± 4.53 17.31 ± 2.45 18.21 ± 2.06  42.54 ± 15.2448.83 ± 5.88 AUC_((0-∞)) (mg · h/L)  65.94 ± 15.55 53.78 ± 8.81 58.69 ±9.91 130.34 ± 34.95 142.55 ± 28.72 Half-Life (h)  1.03 ± 0.19  0.96 ±0.09  1.01 ± 0.31  1.14 ± 0.36  1.51 ± 0.98 Vss (L)  21.4 ± 4.28 23.46 ±2.53 22.36 ± 1.89 22.59 ± 5.24 21.74 ± 3.05 Plasma Clearance 15.75 ±2.90 19.11 ± 3.44 17.39 ± 2.41 16.13 ± 3.33 14.49 ± 2.67 (L/h) Cmax =maximum observed drug concentration; AUC(0-Tlast) = area under the drugconcentration-time curve from time zero to time t last; Vss = apparentvolume of distribution at steady state

Maximum concentrations of Compound I and meropenem were achieved at theend of the 3-hour infusions. Compound I and meropenem exposures (Cmaxand AUC) increased proportionally with dose. The PK parameters ofCompound I and meropenem following a single dose alone or in combinationshow no major changes in the PK properties of either drug (Tables 2 and3). Meropenem PK alone and in combination with Compound I observed inthis study is consistent with published literatures. See, for example,Lodise T. P. et al., “Penetration of meropenem into epithelial liningfluid of patients with ventilator-associated pneumonia,” AntimicrobAgents Chemother. 2011; 55(4):1606-10 and Kuti J. L. et al., “Use ofMonte Carlo simulation to design an optimized pharmacodynamics dosingstrategy for meropenem,” J Clin Pharmacol. 2003; 43(10):1116-23.

Table 4 summarizes the treatment emergent adverse events (AEs) observedin ≧3 subjects receiving the combination of Compound I and meropenem. Nosubjects discontinued due to AEs and no SAEs were observed. There was noevidence of increasing numbers or severity of AEs with increasing doseof either drug alone or in combination, and all AEs were mild ormoderate in severity.

TABLE 4 Treatment Emergent Adverse Events Observed in ≧3 SubjectsReceiving Compound I/Meropenem Compound Compound Compound I CompoundCompound I Pooled Pooled I 250 mg/ I 1000 mg/ 1500 mg/ I 2000 mg/ 2000mg/ Compound Pooled Meropenem Meropenem Meropenem Meropenem MeropenemMeropenem I/ Placebo alone 1 g 1 g 1 g 1 g 2 g Meropenem N (%) (N = 16)(N = 19) (N = 8) (N = 5) (N = 8) (N = 8) (N = 8) (N = 37) Subjects withTEAEs 12 (75%)  18 (95%)  6 (75%)  5 (100%) 5 (63%) 4 (50%) 5 (63%) 25(67%)  Headache 2 (12%) 7 (37%) 3 (37%) 1 (20%) 0 1 (12%) 0 5 (13%) PKcatheter site 2 (12%) 4 (21%) 0 1 (20%) 3 (37%) 0 1 (12%) 5 (13%)hematoma Infusion site pain 2 (12%) 2 (10%) 2 (25%) 0 0 2 (25%) 0 4(11%) PK catheter site pain 3 (19%) 2 (10%) 0 0 1 (12%) 1 (12%) 2 (25%)4 (11%)

Conclusion:

Compound I alone and in combination with 1 or 2 g meropenem was safe andwell-tolerated at all doses tested. AUC and Cmax increasedproportionally with dose and the pharmacokinetic parameters of CompoundI and meropenem are similar. There were no effects of meropenem orCompound I on the PK of the other agent.

Example 3

Example 3 provides a summary of a clinical study of the safety,tolerability and pharmacokinetics of the beta-lactamase inhibitorCompound I alone, meropenem alone, and the combination of both following7 days of TID (three times a day) in healthy adult subjects.

Methods:

Eighty healthy subjects were enrolled into 1 of 5 cohorts in the singleascending dose phase (250 mg, 1000 mg, 1500 mg and 2000 mg Compound I incombination with 1 or 2 g of meropenem). Within each cohort subjectswere administered either Compound I or meropenem on day 1, then werecrossed over to Compound I or meropenem on day 3, then were administeredboth Compound I and meropenem in combination on day 7 followed by 7 daysof TID dosing. All infusions were administered over 3 hours. Intensiveplasma and urine PK sampling was obtained after dosing and assayed usingvalidated HPLC/MS methods. Plasma pharmacokinetics of Compound I aloneand in combination with meropenem after single and 7 days of TID dosingby 3-hour infusions in healthy subjects and plasma pharmacokinetics ofmeropenem alone and in combination with Compound I after single and 7days of TID dosing by 3-hour infusions in healthy subjects areillustrated in FIGS. 5 and 6 respectively.

Results:

The pharmacokinetic parameters, derived using non-compartmental methods,for each drug alone and in combination in the Compound I/meropenem 1 g/1g and 2 g/2 g cohorts are shown in Tables 5 and 6 below. Table 5summarizes Compound I pharmacokinetic parameters (mean±standarddeviation) following a single dose alone (single) and single (first)followed by 7 days of TID dosing (last) of Compound I administered incombination with meropenem as 3-hour infusions to healthy subjects.Table 6 summarizes meropenem pharmacokinetic parameters (mean±standarddeviation) following a single dose alone (single) and single (first)followed by 7 days of TID dosing (last) of meropenem administered incombination with Compound I as 3-hour infusions to healthy subjects.

TABLE 5 Compound I 250 mg Compound I 1000 mg Compound I 1500 mgMeropenem Meropenem Meropenem Alone 1 g Alone 1 g Alone 1 g Single FirstLast Single First Last Single First Last Parameter (N = 16) (N = 8) (N =8) (N = 5) (N = 5) (N = 5) (N = 8) (N = 7) (N = 7) C_(max)  5.20 ± 0.92 5.34 ± 0.78  4.61 ± 0.70 21.98 ± 3.54 23.68 ± 4.38 19.96 ± 1.67 37.23 ±5.33 37.14 ± 4.70 32.74 ± 3.28 (mg/L) AUC_((0-∞)) 17.48 ± 3.02 17.40 ±2.22 14.73 ± 2.19 77.56 ±  81.18 ± 15.38 68.57 ± 8.53 123.66 ± 18.03127.07 ± 20.99 114.32 ± 15.39 (mg · h/L) 15.87 Half-Life  1.18 ± 0.35 1.08 ± 0.21  1.17 ± 0.17  1.56 ± 0.67  1.53 ± 0.32  1.09 ± 0.16  1.21 ±0.24  1.35 ± 0.22  1.08 ± 0.09 (h) Vss (L) 23.15 ± 6.00 22.25 ± 3.0224.92 ± 5.10 21.44 ± 5.22 20.25 ± 3.20 19.93 ± 1.61 19.37 ± 5.14 19.83 ±2.84 18.05 ± 2.22 Plasma 14.69 ± 2.38 14.56 ± 1.76 16.71 ± 2.52 13.35 ±2.83 12.70 ± 2.50 14.55 ± 2.05 12.35 ± 1.75 12.04 ± 1.70 13.12 ± 1.69Clearance (L/h) Compound I 2000 mg Compound I 2000 mg MeropenemMeropenem Alone 1 g Alone 2 g Single First Last Single First LastParameter (N = 8) (N = 8) (N = 7) (N = 8) (N = 8) (N = 8) C_(max) 39.20± 4.29 41.44 ± 4.38 34.93 ± 3.96  51.44 ± 16.16 51.66 ± 7.26  55.61 ±10.96 (mg/L) AUC_((0-∞)) 133.26 ± 20.89 141.02 ± 21.35 112.31 ± 8.56 159.21 ± 44.58 170.44 ± 31.99 190.43 ± 32.90 (mg · h/L) Half-Life (h) 1.31 ± 0.32  1.43 ± 0.22  1.19 ± 0.21  1.39 ± 0.20  1.98 ± 0.81  1.37 ±0.24 Vss (L) 22.02 ± 2.24 22.43 ± 2.00 24.95 ± 2.63 21.37 ± 3.33 21.84 ±3.50 17.50 ± 1.99 Plasma 15.32 ± 2.33 14.44 ± 1.97 17.61 ± 1.44 13.43 ±3.23 12.08 ± 2.09 10.42 ± 1.85 Clearance (L/h) Cmax = maximum observeddrug concentration; AUC(0-Tlast) = area under the drugconcentration-time curve from time zero to time t last; Vss = apparentvolume of distribution at steady state; First—First dose of TID dosingfor 7 days; Last—Last Dose after 7 days of TID dosing

TABLE 6 Meropenem 1 g Compound I Compound I Compound I Alone 250 mgAlone 1000 mg Alone 1500 mg Single First Last Single First Last SingleFirst Last Parameter (N = 16) (N = 8) (N = 8) (N = 9) (N = 5) (N = 5) (N= 13) (N = 7) (N = 7) C_(max) 16.35 ± 3.04 17.17 ± 4.81 15.83 ± 1.9618.93 ± 3.65 20.16 ± 3.97 17.04 ± 1.65 20.75 ± 2.23 20.76 ± 4.53 20.36 ±4.70 (mg/L) AUC_((0-∞)) 51.32 ± 8.88  52.31 ± 47.64 ± 4.91  59.77 ±12.09  65.88 ± 15.33 54.52 ± 6.96 64.97 ± 8.86  65.94 ± 15.55  66.09 ±14.41 (mg · h/L) 12.88 Half-Life  0.98 ± 0.18  0.91 ± 0.14  0.98 ± 0.11 0.96 ± 0.11  1.15 ± 0.21  0.94 ± 0.03  0.89 ± 0.08  1.03 ± 0.19  0.88 ±0.09 (h) Vss (L) 25.86 ± 6.55 22.18 ± 2.63 26.44 ± 4.74 21.59 ± 3.2121.06 ± 4.50 21.19 ± 2.43 18.89 ± 2.62  21.4 ± 4.28 18.53 ± 4.31 Plasma20.04 ± 3.40 16.94 ± 2.47 20.96 ± 2.04 17.39 ± 3.71 15.84 ± 3.57  18.4 ±2.24 15.64 ± 1.98 15.75 ± 2.90 15.59 ± 3.18 Clearance (L/h) Meropenem 1g Meropenem 2 g Compound I Compound I Alone 2000 mg Alone 2000 mg SingleFirst Last Single First Last Parameter (N = 14) (N = 7) (N = 7) (N = 14)(N = 8) (N = 8) C_(max) (mg/L) 17.31 ± 2.45 18.21 ± 2.06 15.81 ± 1.29 42.54 ± 15.24 48.83 ± 5.88 43.35 ± 8.82 AUC_((0-∞)) 53.78 ± 8.81 58.69± 9.91 48.06 ± 2.01 130.34 ± 34.95 142.55 ± 28.72 137.71 ± 26.37 (mg ·h/L) Half-Life (h)  0.96 ± 0.09  1.01 ± 0.31  1.08 ± 0.15  1.14 ± 0.36 1.51 ± 0.98  1.07 ± 0.16 Vss (L) 23.46 ± 2.53 22.36 ± 1.89 24.97 ± 2.4122.59 ± 5.24 21.74 ± 3.05 20.08 ± 3.20 Plasma 19.11 ± 3.44 17.39 ± 2.4120.65 ± 0.84 16.13 ± 3.33 14.49 ± 2.67 14.77 ± 2.84 Clearance (L/h) Cmax= maximum observed drug concentration; AUC(0-Tlast) = area under thedrug concentration-time curve from time zero to time t last; Vss =apparent volume of distribution at steady state; First—First dose of TIDdosing for 7 days; Last—Last Dose after 7 days of TID dosing

Maximum concentrations of Compound I and meropenem were achieved at theend of the 3-hour infusions. Compound I and meropenem exposures (C_(max)and AUC) increased proportionally with dose. The PK parameters ofCompound I and meropenem alone or in combination show no major changesin the PK properties of either drug (see Tables 5 and 6). There was noaccumulation of either Compound I or meropenem observed after 7 days ofTID dosing. Meropenem PK alone and in combination with Compound Iobserved in this study is consistent with published literatures.

Table 7 summarizes the number (%) of subjects with at least onetreatment emergent AE and number of adverse events during the multipledose phase. One subject who received meropenem 1 g/Compound I 2 gdiscontinued early due to an AE of thrombophlebitis. All AEs, except 2were mild or moderate in severity. Mild nausea was observed only in thesubjects who received meropenem 2 g, either alone or in combination.There is no evidence that the addition of Compound I changed the AEprofile of meropenem.

TABLE 7 Number (%) of Subjects with at least one Treatment Emergent AEand Number of Events [ ] during the Multiple Dose Phase Cohort 1 Cohort2 Cohort 3 Cohort 4 Cohort 5 250 mg 1 g 1.5 g 2 g 2 g All Compound ICompound I Compound I Compound I Compound I Pooled Pooled Compound I/Pooled and 1 g and 1 g and 1 g and 1 g and 2 g 2 g Meropenem Meropenem N(%) Placebo Meropenem Meropenem Meropenem Meropenem Meropenem Meropenem(1 and 2 g) Combination [Number of AEs] (N = 18) (N = 8) (N = 5) (N = 8)(N = 8) (N = 8) (N = 5) (N = 21) (N = 45) AEs 15 (83%) 7 (88%) 5 (100%)7 (88%) 7 (88%) 8 (100%) 4 (80%) 20 (95%) 41 (91%) [34]  [17]  [20] [16]  [20]  [34]  [24]  [67] [155]  Moderate or Severe  2 (11%) 2 (25%)0 1 (13%) 4 (50%)  3 (38%) 0  5 (24%) 13 (29%) AEs [2] [2] [1] [5] [3][5] [14]  SAEs 0 0 0 0 0 0 0 0 0

Conclusion:

Compound I alone and in combination with 1 g or 2 g meropenem was safeand well tolerated at all doses tested, with no evidence that the safetyprofile of meropenem was changed by the addition of Compound I. Therewas no accumulation of either Compound I or meropenem observed after 7days of TID dosing. There were no effects of meropenem on thepharmacokinetics of Compound I or vice versa.

Example 4

Example 4 provides a summary of a preliminary study of thepharmacokinetics of the combination of Compound I (2 g) and meropenem (2g) in healthy adult subjects by a 1-hour or 3-hour infusion.

Results:

The pharmacokinetics of Compound I after 3-hour or 1-hour infusions (2 gCompound I alone and in combination with 2 g meropenem) in healthysubjects are illustrated in FIGS. 7 and 8 respectively. The meanpharmacokinetics of Compound I after 1-hour or 3-hour infusions of 2 gCompound I in combination with 2 g meropenem in healthy subjects issummarized in FIG. 9. With respect to Compound I, no effects ofmeropenem on the pharmacokinetics of Compound I were observed witheither infusion rate. In addition, there is no significant effect ofinfusion rate on Compound I exposure (p=0.18).

The pharmacokinetics of meropenem after 3-hour or 1-hour infusions (2 gmeropenem alone and in combination with 2 g Compound I) in healthysubjects are illustrated in FIGS. 10 and 11 respectively. The meanpharmacokinetics of meropenem after 1-hour or 3-hour infusions of 2 gmeropenem in combination with 2 g Compound I in healthy subjects issummarized in FIG. 12. The pharmacokinetics of meropenem open-lactamafter 1-hour infusions of 2 g alone and in combination with 2 g CompoundI and the mean pharmacokinetics of meropenem open-lactam after 1 or3-hour infusions of 2 g meropenem in combination with 2 g Compound I areillustrated in FIGS. 13 and 14.

For meropenem, no effects of Compound I on the pharmacokinetics ofmeropenem were observed with either infusion rate. Meropenem exposure(AUC) after a 3 hour infusion of 2 g meropenem is consistent withpublished literatures. There was an increase in meropenem exposure (AUC)with 1-hour infusion compared to 3-hour infusion. Meropenem exposure(AUC) after a 1 hour infusion of 2 g meropenem is about 48% greater thanthat observed after a 3 hour infusion of 2 g meropenem (211 vs 142mg*h/L). Meropenem weight adjusted clearance (Cl) after a 1 hourinfusion of 2 g meropenem is about 25% slower than that observed after a3 hour infusion (0.14 vs 0.19 l/h/kg; p=0.015). Possible reasons for thedifference observed in meropenem weight adjusted clearance may due tosaturable renal clearance at 2 g dose due to high Cmax or longerinfusion reduces the “dose” due to degradation (opening of the β-lactamring results in formation of meropenem open-lactam).

Example 5

Example 5 provides a summary of an open-label study of the safety andpharmacokinetics of the combination of Compound I and meropenem insubjects with reduced renal function, including patients with standardhemodialysis.

The safety and pharmacokinetics of a single IV dose of 1 g meropenemplus 1 g Compound I, infused over 3 hours, was evaluated. Forty onesubjects were enrolled in 5 groups based on their degree of renalinsufficiency. The five cohorts included: patients with normal renalfunction (CrCl≧90 ml/min), mild renal impairment (CrCl 60-89 ml/min),moderate renal impairment (CrCl 30-<60 ml/min), severe renal impairment(CrCl<30 ml/min, and patients with end stage renal disease requiringhemodialysis. Patients on renal replacement therapy other than standardhemodialysis (including continuous veno-venous hemofiltration,continuous veno-venous hemodialysis and continuous renal replacementtherapy) were not studied.

FIG. 15 shows the relation between estimated GFR and meropenem orCompound I plasma clearance. The plasma clearance of both drugs remainedsimilar throughout the range of renal function as evidenced by theclustering of values and the linear decline in clearance with decreasingrenal function.

The removal of meropenem and Compound I during hemodialysis was studiedin 9 patients with severe renal insufficiency on chronic hemodialysis.Patients received a single meropenem 1 g/Compound I 1 g dose, followedby a hemodialysis session. Both meropenem and Compound I were removedfrom plasma by hemodialysis. These data indicate that maintenance dosesof each drug (adjusted for degree of underlying endogenous renalfunction) should be administered after a dialysis session.

Determination of the Combination of Compound I/Meropenem Dosage inPatients with Renal Impairment

Dosage adjustment according to degree of renal impairment was determinedby analysis of estimates of each subject's pharmacokinetics anddetermining exposures according to possible dosage regimens of meropenemor Compound I. The objective was to maintain exposures (as AUC) acrossthe range of renal function to as consistent as possible across thespectrum of renal function. In view of PK-PD analyses in nonclinicalmodels that show AUC is linked to efficacy for Compound I, AUC was theappropriate controller of efficacy for this agent. Since T>MIC is thePK-PD index important of meropenem, different dosing intervals wereevaluated to insure T>MIC_(breakpoint) was above threshold values(T>MIC>40%) for efficacy. For purposes of this analysis, the forecastedsusceptibility breakpoint for meropenem based on the 2 gram dose and3-hour infusion was 8 μg/ml. Free drug was considered for both meropenemand Compound I (plasma protein binding of 6% and 33%, respectively).

Meropenem

Table A shows meropenem AUC measured in each patient and PK-PD indicesfor three potential dosage regimens in each patient according tomeasured meropenem PK in each subject. Meropenem dosage regimens wereidentified for each of the strata of renal function that would meet orachieve target exposures (T>MIC of at least 40%) in all subjects (seeshaded cells).

Table A summarizes the Analysis of different meropenem dosing regimensby individual subjects. The PK-PD target for meropenem is a T>MIC of atleast 40% of the dosage interval where the MIC is 8 μg/mL. The shadingin different creatinine clearance groups denotes the recommendedmeropenem dosing regimen.

TABLE A Expected Meropenem Time, in hours per day, (% of dosinginterval) Above MIC of 8 μg/ml. According to Dosage Regimen 2 g q8hEstimated Mean Creatinine 13.8 (58) Clearance Range (ml/min) 10.5-16.5500 mg 500 mg Subject Normal (44-69) 1 g q8h 1 g q24h q12h q24h >50mL/min group 4602 83 16.5 (69)   13.5 (56)   4.5 (19)   7 (29) 3.5 (15) 5609 79 15.0 (63)   12 (50)  4 (17) 6 (25)  3 (13) 5607 77 16.5 (69)  13.5 (56)   4.5 (19)   7 (29) 3.5 (15)  5618 77 16.5 (69)   13.5 (56)  4.5 (19)   7.6 (32)   3.8 (16)  4601 71 20 (83)  16.5 (69)   5.5 (23)  8 (33)  4 (17) 5605 67 16.5 (69)   13.5 (56)   4.5 (19)   7 (29) 3.5(15)  5606 56 20 (83)  16.5 (69)   5.5 (23)   9 (38) 4.5 (19)  4613 5520 (83)  16.5 (69)   5.5 (23)   7.6 (32)   3.8 (16)  30-49 ml/min group5603 46 24 (100) 18 (75)  6 (25) 10 (42)   5 (21) 5608 44 24 (100) 18(75)  6 (25) 9 (38) 4.5 (19)  5620 42 24 (100) 16.5 (69)   5.5 (23)   8(33)  4 (17) 5611 40 24 (100) 24 (100) 8 (33) 12 (50)   6 (25) 5610 3824 (100) 24 (100) 10 (42)  12 (50)   6 (25) 5614 32 24 (100) 24 (100) 10(42)  14 (58)   7 (29) 10-19 ml/min group 5616 15 24 (100) 24 (100) 12(50)  20 (83)  10 (42) 5617 14 24 (100) 24 (100) 12 (50)  16 (67)   8(33) 4636 14 24 (100) 24 (100) 10 (42)  16 (67)   8 (33) 5621 12 24(100) 24 (100) 12 (50)  20 (83)  10 (42) 5615 11 24 (100) 24 (100) 12(50)  16 (67)   8 (33) 5612 10 24 (100) 24 (100) 14 (58)  24 (100) 12(50) 5-9 ml/min group 4640 8 24 (100) 24 (100) 24 (100) 24 (100) 12 (50)5633 7 24 (100) 24 (100) 24 (100) 24 (100) 12 (50) 5637 7 24 (100) 24(100) 14 (58)  20 (83)  10 (42) 5642 6 24 (100) 24 (100) 24 (100) 24(100) 12 (50) 5634 6 24 (100) 24 (100) 24 (100) 24 (100) 12 (50) 5641 524 (100) 24 (100) 24 (100) 24 (100)  24 (100) 5638 5 24 (100) 24 (100)24 (100) 24 (100) 12 (50)

Compound I

Table B shows Compound I AUC measured in each patient and 24 h AUC forthree potential dosage regimens according to measured Compound Iclearance in each subject. Since AUC is the target PK metric andCompound I clearance remained close to meropenem clearance, unit and 24hr doses remained at a 1:1 ratio throughout the range of renal function.

Considerations for Subjects with Creatinine Clearance<10 ml/Min

As noted in FIG. 15, as creatinine clearance falls below 10 ml/min,meropenem non-renal clearance assumes a greater proportion of totalclearance. In contrast, Compound I has no measurable non-renalclearance. Thus, to maintain a 1:1 dose ratio to provide therapeuticexposures of each component and to avoid accumulation of Compound I,patients with a creatinine clearance<10 ml/min should receivehemodialysis about every 3 days (i.e., twice weekly).

Table B provides a summary of the analysis of different Compound Idosing regimens by individual subjects enrolled the study. The shadingin different creatinine clearance groups denotes the recommendedmeropenem dosing regimen.

TABLE B EXPECTED COMPOUND I FREE DRUG 24 H AUC (MG * HR/L) ExpectedMeropenem Time, in hours per day, (% of dosing interval) Above MIC of 8μg/ml. According to Dosage Regimen Estimated 2 g q8h Creatinine ObservedMean Clearance AUC_(0-inf) 358 (ml/min) following Range 500 mg 500 mgSubject Normal 1 g dose 284-470 1 g q8h 1 g q24h q12h q24h >50 mL/mingroup 4602 83 70.0 420.0 210.0 70.0 70.0 35.0 5609 79 62.7 376.3 188.262.7 62.7 31.4 5607 77 69.2 415.0 207.5 69.2 69.2 34.6 5618 77 88.4530.5 265.2 88.4 88.4 44.2 4601 71 70.7 424.2 212.1 70.7 70.7 35.4 560567 68.1 408.7 204.3 68.1 68.1 34.1 5606 56 108.4 650.6 325.3 108.4 108.454.2 4613 55 108.0 648.1 324.0 108.0 108.0 54.0 30-49 ml/min group 560346 119.3 715.7 357.8 119.3 119.3 59.6 5608 44 129.1 774.5 387.2 129.1129.1 64.5 5620 42 115.2 690.9 345.5 115.2 115.2 57.6 5611 40 228.11368.4 684.2 228.1 228.1 114.0 5610 38 251.1 1506.5 753.3 251.1 251.1125.5 5614 32 310.6 1863.5 931.8 310.6 310.6 155.3 10-19 ml/min group5616 15 505.1 3030.3 1515.2 505.1 505.1 252.5 5617 14 427.3 2563.81281.9 427.3 427.3 213.7 4636 14 493.6 2961.8 1480.9 493.6 493.6 246.85621 12 790.7 4744.3 2372.2 790.7 790.7 395.4 5615 11 830.3 4981.62490.8 830.3 830.3 415.1 5612 10 719.6 4317.6 2158.8 719.6 719.6 359.85-9 ml/min group 4640 8 8617.7 51706.2 25853.1 8617.7 8617.7 4308.9 56337 4189.5 25137.0 12568.5 4189.5 4189.5 2094.8 5637 7 794.5 4767.0 2383.5794.5 794.5 397.3 5642 6 923.2 5539.8 2769.9 923.2 923.2 461.7 5634 6840.0 5040.0 2520.0 840.0 840.0 420.0 5641 5 7581.7 45490.2 22745.17581.7 7581.7 3790.0 5638 5 2289.0 13734.0 3270.0 2289.0 2289.0 1144.5

Based on the above analysis, the Compound I/meropenem Combination dosageregimens in Table C can be used for subjects with impaired renalfunction.

TABLE C COMPOUND I/MEROPENEM COMBINATION DOSAGE ACCORDING TO RENALFUNCTION Estimated Creatinine the Combination Dosage Regimen Clearance(ml/min) (All doses infused over 3 hrs) ≧50 Meropenem 2 g/Compound I 2 gq8 h ≧30-49 Meropenem 1 g/Compound I 1 g q8 h ≧20-29 Meropenem 1g/Compound I 1 g q12 h ≧10-19 Meropenem 500 mg/Compound I 500 mg q 12 h <10 Meropenem 500 mg/Compound I 500 mg every q 24 h¹ ¹Dosage regimenassumes patients receive hemodialysis at least twice per week.Maintenance doses of the Combination in these patients should beadministered as soon as possible after the dialysis session. Forexample, if a subject is scheduled to receive the Combination at 18:00but receives hemodialysis at 13:00, the planned 18:00 Combination doseshould be given after the dialysis session is completed (rather thanwaiting until 18:00).

It is concluded that dose adjustment for renal function can be based oneither meropenem or Compound I as both drugs are affected similarly asrenal function declines. For subjects with creatinine clearance of equalor greater than 50 ml/min, there is no need for dose adjustment. Thestandard dosage of 2 g Compound I/2 g meropenem TID (every 8 hours) canbe used. For subjects with creatinine clearance of equal or greater than30 ml/min and less than 50 ml/min, a reduced dosage of 1 g Compound I/1g meropenem TID (every 8 hours) can be used and still achieve desiredeffects. For subjects with creatinine clearance of equal or greater than20 ml/min and less than 30 ml/min, a reduced dosage of 1 g Compound I/1g meropenem administered every 12 hours can be used. For subjects withcreatinine clearance of equal or greater than 10 ml/min and less than 20ml/min, a reduced dosage of 500 mg Compound I/500 mg meropenemadministered every 12 hours can be used. For subjects with creatinineclearance of less than 10 ml/min, a reduced dosage of 500 mg CompoundI/500 mg meropenem every 24 hours can be used.

Example 6

Example 6 provides a summary of a randomized, open-label clinical studyevaluating the plasma, epithelial lining fluid (ELF), and alveolarmacrophage (AM) concentrations of the combination of 2 g Compound I/2 gmeropenem (“the Combination”) in healthy adult subjects.

For lower respiratory tract infections, epithelial lining fluid (ELF)and alveolar macrophages (AM) have been advocated as important infectionsites for common extracellular and intracellular pathogens,respectively. Studies with bronchoscopy and bronchoalveolar lavage(BAL), which can reliably assess the intrapulmonary penetration ofantibiotics into the ELF and AM, are needed. The primary objectives ofthis pharmacokinetic study are to determine and compare the plasma, ELF,and AM concentrations of Compound I and meropenem administered followingmultiple intravenous doses (2 g meropenem/2 g Compound I administeredq8h for 3 doses) in healthy male and female adult subjects. A secondaryobjective of this study was to assess the safety and tolerability ofintravenous administration of the Combination in healthy adult subjects.

Methods for Pharmacokinetic Analysis

Study Design and Subjects.

A total of twenty-five (n=25) male and female subjects who met the studyentry criteria and completed all phases of the pharmacokinetic studywere included in this pharmacokinetic analysis. Each subject receivedthe Combination (2 g of meropenem/2 g of Compound I) administered every8 hours for a total of three doses under direct observation at the studysite. Blood samples were collected to measure drug concentrations inplasma prior to (time 0), and at 1.5, 2.95, 3.083, 3.25, 3.5, 4, 6, and8 hours after the start of a 3-hour intravenous infusion of the thirdcombination dose. Each subject had a single standardized bronchoscopywith BAL scheduled at a timed interval following the last dose of theCombination as indicated in the following table:

BAL Sampling Times after Start of the Third Infusion of the CombinationSampling Time 1.5 h 3.25 h 4 h 6 h 8 h Subjects (n) 5 5 5 5 5

Urea has been commonly used as an endogenous marker to estimate theapparent volume of ELF. Blood samples to determine plasma ureaconcentrations were obtained just prior to scheduled bronchoscopy.Aliquots of BAL were obtained to determine urea concentrations in BALand cell count with differential. The standardized bronchoscopy with BALprocedure for the collection of intrapulmonary samples has beenpreviously described in the references listed below.

Drug and Urea Assays.

Sample preparation procedures and assays for meropenem, Compound I, andmeropenem open-lactam concentrations in plasma, ELF, and AM wereperformed with a high-performance liquid chromatography with massspectrometric detection at MicroConstants, Inc., San Diego, Calif.(Reports MC14B-0013, MC14B-0015, MC14I-011, and MC14I-0012). The ureaconcentrations in plasma and BAL were performed with a microplate-basedmethod with an O-phthalaldehyde chromogenic solution at MicroConstants,Inc., San Diego, Calif.

Pharmacokinetic Calculations of Plasma Concentrations.

Noncompartmental methods were used to generate pharmacokineticparameters for meropenem, Compound I, and meropenem open-lactam inplasma. Peak plasma concentration (C_(max)) and time to C_(max)(T_(max)) were read from the observed plasma concentration-time profileafter the start of the intravenous infusion of the third Combinationdose. Area under the plasma concentration-time curve over 8 hours(AUC₀₋₈) after the third dose was calculated with the linear-logtrapezoidal rule (WinNonlin®, version 6.3, Pharsight Corporation, Cary,N.C.). The elimination rate constant (β) was determined by nonlinearleast-squares regression. Elimination half-life (t_(1/2)) was calculatedby dividing β into the natural logarithm of two. For meropenem andCompound I, the apparent clearance (CL) and volume of distribution terms(V_(ss)) were calculated with the standard noncompartmental equationsembedded in the WinNonlin® program.

Calculations of ELF Volume and Antibiotic Concentrations in ELF and AM.

The calculations of ELF volume and drug concentrations in ELF and AMwere performed with BAL supernatant and pulmonary (alveolar) cells(“cell pellet”) from aspirates recovered from the 2^(nd), 3^(rd), and4^(th) instillations (BAL2). The concentration of drug (ABX_(ELF)) inthe epithelial lining fluid (ELF) was determined as follows:

ABX _(ELF) =ABX _(BAL)×(V _(BAL) /V _(ELF))

where ABX_(BAL) is the measured concentration of meropenem, Compound Ior meropenem open-lactam in BAL fluid, V_(BAL) is the volume ofaspirated BAL fluid, and V_(ELF) is the volume of ELF sampled by theBAL. V_(ELF) is derived from the following:

V _(ELF) =V _(BAL)×Urea_(BAL)/Urea_(P)

where Urea_(BAL) is the concentration of urea in BAL fluid and Urea_(P)is the concentration of urea in plasma.

The concentration of drug (ABX_(AM)) in the alveolar cells (AC) wasdetermined as follows:

ABX _(AM) =ABX _(M) /V _(AC)

where ABX_(M) is the measured concentration of meropenem, Compound I ormeropenem open-lactam in the 1-ml cell suspension, and V_(AC) is thevolume of alveolar cells in the 1-ml cell suspension. Differential cellcount was performed to determine the number of macrophages present. Amean macrophage cell volume of 2.42 μl/106 cells was used in thecalculations for volume of alveolar cells in the pellet suspension.

The concentration ratios of ELF and AM to the simultaneous plasmaconcentrations were calculated for each subject and summarized for eachgroup at each sampling time. The mean and median concentrations ofmeropenem and Compound I from the bronchopulmonary sampling times (e.g.,1.5, 3.25, 4, 6, and 8 hours) were used to estimate the AUC₀₋₈ ofplasma, ELF, and AM. The 8-hour sampling time was also used as a valueat time zero for determining the area term of plasma, ELF, and AM. TheAUC₀₋₈ for each matrix was determined with the linear trapezoidalmethod. The ratio of AUC₀₋₈ of ELF to plasma and AM to plasma werecalculated.

Results

Twenty-six (26) healthy adult subjects were enrolled into this study.One subject was discontinued from the study due to adverse events andpharmacokinetic phases (e.g., blood sample collection to measure drugconcentrations in plasma and a bronchoscopy with BAL at the scheduledsampling time [4-hour]) were not performed. The characteristics of the25 study subjects receiving the Combination for three doses andcompleting all phases of the pharmacokinetic study are reported in Table8.

Mean (±SD) plasma concentrations of meropenem before and after the startof the intravenous infusion of the third Combination dose are displayedin FIG. 16. The mean (±SD) C_(max) and AUC₀₋₈ for plasma meropenemconcentrations were 58.2±10.8 μg/mL and 185.5±33.6 μg·h/mL,respectively. The mean (±SD) pharmacokinetic parameters of meropenem inplasma are summarized in Table 9. Mean (±SD) plasma concentrations ofCompound I before and after the start of the intravenous infusion of thethird Combination dose are displayed in FIG. 17. The mean (±SD) C_(max)and AUC₀₋₈ for plasma Compound I concentrations were 59.0±8.4 μg/mL and204.2±34.6 μg·h/mL, respectively. The mean (±SD) pharmacokineticparameters of Compound 1 in plasma are summarized in Table 10.

The mean (±SD) concentrations of meropenem in plasma and ELF at thebronchopulmonary sampling times are illustrated in FIG. 18. The meanconcentrations of meropenem in plasma and ELF ranged from 1.36 to 41.2μg/mL and 2.51 to 28.3 μg/mL, respectively. The mean (±SD)concentrations of meropenem after the last dose in plasma, ELF, and AMat the five bronchopulmonary sampling times are reported in Table 11.The concentrations of meropenem in the alveolar cells were below thequantifiable limit for all samples.

The mean (±SD) concentrations of Compound I in plasma, ELF, and AM atthe bronchopulmonary sampling times are illustrated in FIG. 19. The meanconcentrations of Compound I in plasma and ELF ranged from 2.74 to 51.1μg/mL and 2.61 to 26.1 μg/mL, respectively. FIGS. 20 and 21 illustratethe similar magnitude and time course of concentrations for meropenemand Compound I in plasma and ELF. The mean (±SD) concentrations ofCompound I after the last dose in plasma, ELF, and AM at the fivebronchopulmonary sampling times are reported in Table 12. Alveolarmacrophage concentrations of Compound I were measurable for all samplesand ranged from 1.26 to 93.9 μg/mL.

The mean (±SD) ratios of ELF to the simultaneous plasma concentrationsfor meropenem are reported in Table 13. The mean ratios of ELF tosimultaneous plasma concentrations for meropenem during the 8-hourperiod after drug administration ranged from 0.525 to 2.13. The AUC₀₋₈values based on mean and median ELF concentrations were 111.7 and 102.4μg·h/mL, respectively. The ratio of ELF to total plasma meropenemconcentrations based on the mean and median AUC₀₋₈ values were 0.63 and0.58, respectively. The ratios of ELF to unbound plasma meropenemconcentrations (protein binding=2%) based on the mean and median AUC₀₋₈values were 0.65 and 0.59, respectively.

The mean (±SD) ratios of ELF and AM to the simultaneous plasmaconcentrations for Compound I are reported in Table 14. The mean ratiosof ELF and AM to simultaneous plasma concentration for Compound I duringthe 8-hour period after drug administration ranged from 0.45 to 1.01 and0.062 to 2.58, respectively. The AUC₀₋₈ values based on mean and medianELF concentrations were 105.1 and 96.7 μg·hr/mL, respectively. The ratioof ELF to total plasma Compound I concentrations based on the mean andmedian AUC₀₋₈ values were 0.53 and 0.48, respectively. The ratios of ELFto unbound plasma Compound I concentrations (protein binding=33%) basedon the mean and median AUC₀₋₈ values were 0.79 and 0.72, respectively.

SUMMARY

The Combination (2 g meropenem/2 g Compound I) administered every 8hours, as 3-hour IV infusions, achieved a similar time course andmagnitude of meropenem and Compound I concentrations in plasma and ELF.The intrapulmonary penetration of meropenem and Compound I based onAUC₀₋₈ values of ELF and total plasma concentrations were approximately63% and 53%, respectively. When unbound plasma concentrations wereconsidered, penetration was 65% and 79% for meropenem and Compound I,respectively. Results from this study lend support to exploring themeropenem 2 g/Compound I 2 g combination as a potential antimicrobialagent for the treatment of lower respiratory tract bacterial infectionscaused by susceptible pathogens.

The concentrations of meropenem in the alveolar cells were below thequantifiable limit for all samples. In contrast, concentrations ofCompound I were measurable for all alveolar cell samples and AMconcentrations ranged from 1.26 to 93.9 μg/mL. It is worth noting thattwo subjects of the 6-hour sampling time had the highest reportedconcentrations of Compound I in AM (35.4 and 93.9 μg/mL) whichconsequently inflated the mean ratio of AM to plasma concentration(2.58±3.57, Table 14). Both of these subjects had extremely highconcentrations of red blood cells in their BAL fluid (176,000 and226,250 cells/mm³) which may have contributed to such high measurementsof AM concentrations.

The ratio of systematic exposure of meropenem open-lactam to meropenemwas approximately 11% and 15% based on comparison of maximum plasmaconcentration and AUC₀₋₈ values, respectively. The mean ELFconcentrations of meropenem open-lactam ranged from only 1.81 to 2.69μg/mL during the first 6 hours after meropenem administration, and allELF concentrations of meropenem open-lactam were below the quantifiablelimit at the 8-hour sampling time. Only three AM concentrations ofmeropenem open-lactam were measurable and ranged from 1.91 to 8.46μg/mL.

Conte et al. administered meropenem at a dose of 500 mg, 1 gram or 2gram every 8 hours, as 30-minute IV infusions, for a total of fourdoses. The mean meropenem ELF concentrations at 1, 2, 3, 5, and 8 hourswere 5.3, 2.7, 1.9, 0.7, and 0.2 μg/mL for the 500 mg dose and 7.7, 4.0,1.7, 0.8, and 0.03 μg/mL for the 1 gram dose. The ratios of ELFconcentrations to total plasma concentrations at the sampling timesranged from 0.49 to 2.3 for the 500 mg dose and 0.32 to 0.53 for the 1gram dose. The intrapulmonary penetration of meropenem based on AUC₀₋₈values of ELF and total plasma concentrations were approximately 43% and28% for the 500 mg and 1 gram doses, respectively. For the 2 gram dose,the mean meropenem ELF concentrations and penetration ratios at 1- and3-hour sampling times were 2.9 and 2.8 μg/mL, and 0.05 and 0.22,respectively. For the 2 gram dose, the number of observations werelimited (n=8) and calculations of AUC₀₋₈ value for ELF was not possible.

The meropenem findings in this study are not directly comparable tothose of Conte et al due to differences in study design. This studyevaluated a 2 gram dose of meropenem administered as a prolongedinfusion of 3 hours and in combination with Compound I. In addition,this study included more extensive collection of ELF concentrations(n=30) during the 8-hour dosing interval which allowed an accurateestimation of AUC₀₋₈ value. Higher mean concentrations of meropenem inplasma and ELF after 2 gram administration with prolonged infusions(range: 1.36 to 41.2 μg/mL and 2.51 to 28.3 μg/mL, respectively) wasobserved. It is also possible that more prolonged infusions ofcarbapenems may provide higher penetration into ELF, as has beenreported previously for biapenem (Kikuchi et al). The mean ratios of ELFto simultaneous plasma concentrations for meropenem during the 8-hourperiod ranged from 0.525 to 2.13. The AUC₀₋₈ values based on mean andmedian ELF concentrations were 111.7 and 102.4 μg·h/mL, respectively.The ratio of ELF to total plasma meropenem concentrations based on themean and median AUC₀₋₈ values were 0.63 and 0.58, respectively. Thesedata support further study of the Compound I/meropenem combination fortreatment of pulmonary infections.

TABLE 8 CHARACTERISTICS OF STUDY SUBJECTS RECEIVING THE COMBINATIONEVERY 8 HOURS FOR 3 DOSES BAL Total Cell Count Sampling Age HeightWeight BMI in BAL Fluid Macrophages Time Sex (years) (cm) (kilograms)(kg/m²) (mm³) (%) 1.5-hour   5 M 32 ± 9 181 ± 7 83.2 ± 5.5  25.5 ± 3.3114 ± 46 89 ± 7 3.25-hour   3 M, 2 F  40 ± 12  174 ± 10 80.5 ± 11.9 26.6± 1.6  92 ± 52  83 ± 13 4-hour 5 M 40 ± 9  179 ± 10 80.5 ± 13.0 25.2 ±2.3 173 ± 80 91 ± 4 6-hour 3 M, 2 F 43 ± 8 169 ± 9 80.9 ± 8.3  28.5 ±0.7  197 ± 186  80 ± 10 8-hour 2 M, 3 F  40 ± 12 168 ± 5 76.2 ± 9.2 26.9 ± 2.1 130 ± 76 85 ± 8 Data are expressed as mean ± SD except forsex M = males; F = females BMI = body mass index = weight [kg] ÷ (height[m])²

TABLE 9 NONCOMPARTMENTAL PHARMACOKINETICS PARAMETERS IN PLASMA OFMEROPENEM 2 G EVERY 8 HOURS FOR 3 DOSES C_(max) T_(max) AUC₀₋₈ t_(1/2)V_(ss) CL (μg/mL) (hours) (μg · hr/mL) (hours) (Liters) (L/hr) AllSubjects^(a) 58.2 ± 10.8 2.98 ± 0.06 185.5 ± 33.6 1.03 ± 0.15 16.3 ± 2.611.1 ± 2.1 1.5-hour BAL Sampling Group^(b) 56.9 ± 19.3 2.95 ± 0.01 167.8± 41.7 0.98 ± 0.05 17.5 ± 2.5 12.5 ± 2.8 3.25-hour BAL SamplingGroup^(b) 57.9 ± 7.5  3.00 ± 0.07 183.8 ± 29.7 1.04 ± 0.13 16.7 ± 2.611.1 ± 1.8 4-hour BAL Sampling Group^(b) 59.6 ± 7.4  2.98 ± 0.06 196.2 ±33.5 1.07 ± 0.15 15.3 ± 2.1 10.5 ± 1.9 6-hour BAL Sampling Group^(b)59.4 ± 11.5 2.98 ± 0.06 197.4 ± 38.7 1.12 ± 0.24 16.1 ± 3.4 10.4 ± 2.08-hour BAL Sampling Group^(b) 57.3 ± 9.0  2.98 ± 0.06 182.4 ± 28.7 0.96± 0.13 15.6 ± 2.8 11.2 ± 1.9 Data are expressed as mean ± SD. ^(a)25subjects per parameter estimate ^(b)5 subjects per parameter estimate

TABLE 10 NONCOMPARTMENTAL PHARMACOKINETICS PARAMETERS IN PLASMA OFCOMPOUND 12 G EVERY 8 HOURS FOR 3 DOSES C_(max) T_(max) AUC₀₋₈ t_(1/2)V_(ss) CL (μg/mL) (hours) (μg · hr/mL) (hours) (Liters) (L/hr) AllSubjects^(a) 59.0 ± 8.4 2.98 ± 0.06 204.2 ± 34.6 1.27 ± 0.21 17.6 ± 2.610.1 ± 1.9 1.5-hour BAL Sampling Group^(b)  56.1 ± 13.0 2.95 ± 0.01183.6 ± 38.6 1.18 ± 0.08 18.6 ± 2.3 11.3 ± 2.6 3.25-hour BAL SamplingGroup^(b) 59.7 ± 5.8 3.00 ± 0.07 210.5 ± 32.2 1.26 ± 0.23 17.3 ± 2.3 9.7 ± 1.6 4-hour BAL Sampling Group^(b) 60.1 ± 5.7 2.98 ± 0.06 213.7 ±35.4 1.34 ± 0.26 16.9 ± 0.9  9.5 ± 1.3 6-hour BAL Sampling Group^(b)60.9 ± 9.7 3.00 ± 0.07 215.8 ± 33.7 1.37 ± 0.27 18.1 ± 3.9  9.5 ± 1.68-hour BAL Sampling Group^(b) 57.9 ± 8.8 2.98 ± 0.06 197.5 ± 36.6 1.18 ±0.16 17.0 ± 3.3 10.4 ± 2.0 Data are expressed as mean ± SD. ^(a)25subjects per parameter estimate ^(b)5 subjects per parameter estimate

TABLE 11 MEROPENEM CONCENTRATIONS IN PLASMA, ELF, AND AM AT TIME OFBRONCHOSCOPY AND BAL Plasma ELF AM BAL Sampling Time (μg/mL) (μg/mL)(μg/mL) 1.5-hour   41.2 ± 5.0 21.4 ± 4.0 BQL 3.25-hour   47.7 ± 7.3 28.3± 6.7 BQL 4-hour 23.8 ± 4.3 16.1 ± 4.8 BQL 6-hour  7.24 ± 2.79  7.52 ±5.29 BQL 8-hour  1.36 ± 0.51  2.51 ± 1.13 BQL Data are expressed as mean± SD 5 subjects per sampling period BQL = below quantifiable limit

TABLE 12 COMPOUND I CONCENTRATIONS IN PLASMA, ELF, AND AM AT TIME OFBRONCHOSCOPY AND BAL Plasma ELF AM BAL Sampling Time (μg/mL) (μg/mL)(μg/mL) 1.5-hour   42.1 ± 5.0 18.6 ± 3.8 2.71 ± 1.44 3.25-hour   51.1 ±6.8 26.1 ± 1.1 8.79 ± 9.43 4-hour 28.2 ± 5.3 15.7 ± 3.4 5.51 ± 3.156-hour 10.8 ± 2.8  8.03 ± 5.80 27.6 ± 39.6 8-hour  2.74 ± 1.12  2.61 ±1.35 4.40 ± 4.10 Data are expressed as mean ± SD 5 subjects per samplingperiod

TABLE 13 RATIOS OF ELF TO TOTAL PLASMA CONCENTRATIONS OF MEROPENEM BALSampling Time ELF to Plasma 1.5-hour   0.525 ± 0.107 3.25-hour   0.590 ±0.079 4-hour 0.705 ± 0.302 6-hour 1.037 ± 0.475 8-hour 2.133 ± 1.366Data are expressed as mean ± SD 5 subjects per sampling period

TABLE 14 RATIOS OF ELF AND AM TO TOTAL PLASMA CONCENTRATIONS OF COMPOUNDI BAL Sampling Time ELF to Plasma AM to Plasma 1.5-hour   0.450 ± 0.1230.062 ± 0.029 3.25-hour   0.508 ± 0.096 0.165 ± 0.163 4-hour 0.570 ±0.159 0.191 ± 0.101 6-hour 0.705 ± 0.329 2.58 ± 3.57 8-hour 1.009 ±0.391 1.603 ± 1.103 Data are expressed as mean ± SD 5 subjects persampling period

Example 7

Example 7 provides a summary of a Hollow-Fiber Model study of thepharmacokinetic profiles of the combination of Compound I and meropenemin two different dosing regimens (2 g meropenem/2 g Compound I and 1 gmeropenem/1 g Compound I) given every 8 hours by 3-hour infusion. Thecombination is highly active against gram-negative pathogens, includingKPC-producing, carbapenem-resistant Enterobacteriaceae K. pneumonia andP. aeruginosa. The objective of this study was to demonstrate theefficacy of meropenem in combination with Compound I against clinicalisolates of P. aeruginosa using simulated human exposures in an in vitrohollow fiber model. The pharmacokinetics simulation was based on datafrom the clinical study disclosed in Example 2.

Methods: Three P. aeruginosa strains were tested. The minimal inhibitoryconcentrations (MICs) were determined by broth microdilution assay usingto CLSI reference methods and are shown in Table D.

TABLE D Bacterial Strains Used In These Studies Meropenem Meropenem (w/8mg/L Compound I) Strain MIC (mg/L) MIC (mg/L) P. aeruginosa PAM3210 2 2P. aeruginosa PAM3377 4-8 4-8 P. aeruginosa PAM3353 8 8

In Vitro PK-PD Model: Six medium sized hollow-fiber cartridges(FiberCell Systems) were used per experiment. Three strains studied induplicate were used for each experiment. Log-phase cells were inoculatedand incubated for 2 hours prior to the start of treatment to achieveabout 10⁸ CFU/mL. Target PK parameters are listed in Tables E and F. Theexposures were based on the published literatures disclosed in Example2. Samples were collected from the central compartment for thedetermination of drug concentrations over a 32 hour period and wereanalyzed using an LC-MS/MS method.

TABLE E Meropenem Pharmacokinetic Parameters Meropenem Average MeropenemPK Parameters Target Actual Half-Life (hrs) 1.33 1.3 Cmax (mg/L) 39 33.9AUC (mg*h/L) 140 129.0

TABLE F The Combination Compound I/Meropenem Pharmacokinetic ParametersAverage Average Meropenem Meropenem Compound I Compound I PK ParametersTarget Actual Target Actual Half-Life (hrs) 1.33 1.4 1.52 1.5 Cmax(mg/L) 39 33.5 30 26.4 AUC (mg*h/L) 140 131.5 106 105.3

Klebsiella pneumoniae carbapenemase (KPC)-producing strains ofEnterobacteriaceae with meropenem alone MIC ranging from 8 to 512 μg/mland with meropenem/Compound I (wherein Compound I was administered atfixed concentration of 8 μg/ml with meropenem, the MIC meropenem rangesfrom ≦0.06 to 8 μg/ml) as well as P. aeruginosa strains with meropenemand meropenem/Compound I MIC 2-8 μg/ml were used.

Results:

Exposure from the combination of 1 g meropenem and 1 g Compound I dosingregimen was associated with effective killing and no regrowth at 32hours of KPC-producing strains of K. pneumonia with meropenem alone (MICranging from 8 to 64 μg/ml) and with the combination of meropenem andCompound I (where Compound I was administered at the fixed concentrationof 4 μg/ml with meropenem, the MIC of meropenem ranges from ≦0.06 to 2μg/ml) (see FIG. 22 and FIG. 23). Several clones of the strains KP1061,KP1087, KP1004 and KP1074 that survived at 32 hours were tested forsusceptibility to meropenem and meropenem/Compound I combination andwere found to be indistinguishable from the pre-exposed strains.

On the other hand, less killing was observed for the strain KP1099 withmeropenem alone (MIC is 128 μg/ml) and the combination of meropenem andCompound I (when Compound I was administered at the fixed concentrationof 4 μg/ml, the MIC of meropenem reduced to 4 μg/ml). See FIG. 23.Regrowth was observed after 16 hours from the start of treatment. Whencolonies of KP1099 that survived exposure to three doses of 1 gmeropenem/1 g Compound I were investigated, their susceptibility tomeropenem/Compound I was reduced 16-32-fold indicating selection ofresistance under the conditions of inadequate exposure.

Importantly, exposure from 2 g meropenem/2 g Compound I dosing regimenwas associated with efficient killing and no regrowth/resistancedevelopment using strains with meropenem alone and meropenem/Compound I.For the strain KP1094, MIC for meropenem alone was as high as 512 μg/ml.However, when Compound I was administered at the fixed concentration of8 μg/ml with meropenem, the observed MIC of meropenem was reduced to 8μg/ml (see FIG. 24).

Exposure from 1 g meropenem/1 g Compound I dosing regimen resulted ineffective killing and no regrowth at 32 hours due to resistancedevelopment for the strain of P. aeruginosa PAM3210 with meropenem andmeropenem/Compound I (when Compound I was administered at the fixedconcentration of 4 μg/ml or 8 μg/ml, the MIC of meropenem remains 2μg/ml. However, regrowth and resistance development occurred in thestrains PAM3353 and PAM3377 with an MIC of 8 μg/ml for meropenem (seeFIG. 25).

For the efficacy of simulated human exposures of meropenem compared tothe combination of Compound I 2 g/meropenem 2 g against Pseudomonasaeruginosa in the in vitro hollow fiber model, it was observed that themodel effectively simulated human exposures of both meropenem andCompound I. (See FIG. 26). Antibacterial activity of meropenem in themodel is shown in FIG. 27. Meropenem 2 g q8h by 3 hour infusion producedover 4 logs of bacterial killing against the strain with an MIC of 2mg/L, almost 4 logs of killing against the strain with an MIC of 4-8mg/L. Resistance developed in the strain with an MIC of 8 mg/L.Antibacterial activity of the combination of 2 g meropenem/2 g CompoundI in the model is shown in FIG. 28. The combination produced over 4 logsof bacterial killing against all strains tested with no regrowth orresistance development over the 32 hour test period. 2 g meropenem/2 gCompound I dosing regimen was efficacious against all three strains. Noresistant mutants were identified among surviving bacterial (see FIG.28). The results are summarized in Table G below.

TABLE G Change in Log CFU MIC Human Equivalent over 32 μg/mL DosageRegimen hours P. aeruginosa PAM3210 Meropenem 2 2 g q8 h by 3 hour >4infusion Meropenem/Compound I 2 2 g/2 g q8 h by 3 hour >4 infusion P.aeruginosa PAM3377 Meropenem 4-8 2 g q8 h by 3 hour 3.7 infusionMeropenem/Compound I 4-8 2 g/2 g q8 h by 3 hour >4 infusion P.aeruginosa PAM3353 Meropenem 8 2 g q8 h by 3 hour 1.3* infusionMeropenem/Compound I 8 2 g/2 g q8 h by 3 hour >4 infusion *ResistanceDeveloped

In conclusion, the PK/PD studies in in vitro models of infectionsdemonstrate that the human exposures from 2 g/2 g combination ofmeropenem/Compound I are associated with extensive killing of targetpathogens and prevention of resistance for the strains with Compound Iat fixed 8 μg/ml and meropenem MIC less or equal to 8 μg/ml. Inaddition, the 2 g/2 g dose combination reduced exposures that areassociated with resistance development.

In addition, the combination of Compound I 2 g/meropenem 2 gadministered every 8 hours by three hour infusion was highly efficaciousin this in vitro model against P. aeruginosa strains with MICs as highas 8 mg/L, with no regrowth and no resistance development over thecourse of the 32 hour study. Meropenem 2 g q8h by 3 hour infusion waseffective against 2 out of 3 strains, but resistance developed in thethird strain with an MIC of 8 mg/L.

1. A method of treating or ameliorating a bacterial infection,comprising administering an effective amount of Compound I or apharmaceutically acceptable salt thereof and meropenem to a subject inneed thereof:

wherein the amount of Compound I or the pharmaceutically acceptable saltthereof is from about 1.0 g to about 3.0 g and the amount of meropenemis from about 1.0 g to about 3.0 g.
 2. The method of claim 1, whereinthe amount of Compound I or the pharmaceutically acceptable salt thereofis about 2.0 g.
 3. The method of claim 1, wherein the amount ofmeropenem is about 2.0 g.
 4. (canceled)
 5. The method of claim 1,wherein Compound I or the pharmaceutically acceptable salt thereof andmeropenem are administered at least once per day.
 6. (canceled)
 7. Themethod of claim 1, wherein the daily dose of Compound I or thepharmaceutically acceptable salt thereof is about 6.0 g and wherein thedaily dose of meropenem is about 6.0 g.
 8. The method of claim 1,wherein the administration is by intravenous infusion.
 9. (canceled) 10.(canceled)
 11. (canceled)
 12. (canceled)
 13. (canceled)
 14. The methodof claim 1, further comprises administering an additional medicamentselected from an antibacterial agent, antifungal agent, an antiviralagent, an anti-inflammatory agent, or an anti-allergic agent.
 15. Amethod of treating or ameliorating a bacterial infection, comprisingselecting for treatment a subject in need for treatment of a bacterialinfection who is suffering from reduced renal function; administering aneffective amount of compound I or a pharmaceutically acceptable saltthereof and meropenem to said subject.


16. The method of claim 15, wherein said subject has a creatinineclearance of ≧30 ml/min and <50 ml/min.
 17. The method of claim 15,wherein said subject has a creatinine clearance of ≧20 ml/min and <30ml/min.
 18. The method of claim 15, wherein said subject has acreatinine clearance of ≧10 ml/min and <20 ml/min.
 19. The method ofclaim 15, wherein said subject has a creatinine clearance of <10 ml/min.20. (canceled)
 21. (canceled)
 22. The method of claim 15, whereinCompound I or the pharmaceutically acceptable salt thereof isadministered in a dose of about 500 mg to about 1.0 g.
 23. (canceled)24. (canceled)
 25. (canceled)
 26. The method of claim 15, whereinmeropenem is administered in a dose of about 500 mg to about 1.0 g. 27.(canceled)
 28. (canceled)
 29. (canceled)
 30. (canceled)
 31. The methodof claim 15, wherein Compound I or the pharmaceutically acceptable saltthereof and meropenem are administered at least once per day. 32.(canceled)
 33. (canceled)
 34. (canceled)
 35. (canceled)
 36. (canceled)37. (canceled)
 38. The method of claim 15, wherein the administration isby intravenous infusion.
 39. (canceled)
 40. (canceled)
 41. (canceled)42. (canceled)
 43. (canceled)
 44. A method of treating or ameliorating alower respiratory tract infection, comprising administering an effectiveamount of Compound I or a pharmaceutically acceptable salt thereof andmeropenem to a subject in need thereof:


45. (canceled)
 46. The method of claim 44, wherein Compound I or thepharmaceutically acceptable salt thereof is administered in a dose rangefrom about 1.0 g to about 3.0 g.
 47. (canceled)
 48. The method of claim44, wherein meropenem is administered in a dose range from about 1.0 gto about 3.0 g.
 49. The method of claim 44, wherein both Compound I orthe pharmaceutically acceptable salt thereof and meropenem areadministered in a dose of about 2.0 g.
 50. The method of claim 44,wherein Compound I or the pharmaceutically acceptable salt thereof andmeropenem are administered at least once per day.
 51. (canceled)
 52. Themethod of claim 44, wherein the daily dose of Compound I or thepharmaceutically acceptable salt thereof is from about 3.0 g to about6.0 g and wherein the daily dose of meropenem is from about 3.0 g toabout 6.0 g.
 53. The method of any one of claim 44, wherein theadministration is by intravenous infusion.
 54. (canceled)
 55. (canceled)56. (canceled)
 57. (canceled)
 58. (canceled)
 59. The method of claim 44,wherein the subject is suffered from infections caused byenterobacteriaceae.